Antimicrobial compounds and methods of use

ABSTRACT

The present disclosure provides antimicrobial compounds, compositions comprising such antimicrobial compounds, and methods of their use, in particular, antibacterial compounds and antifungal compounds. In certain aspects, the antimicrobial compounds are effective against pathogens of hospital-acquired infections. In certain aspects, the antimicrobial compounds are effective against pathogens that are resistant to antibiotics. The antimicrobial compounds can be used in antibacterial compositions, antifungal compositions, antiseptic compositions and disinfectant compositions. The antimicrobial compounds can be used as adjuncts in antibacterial compositions and antifungal compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119 of U.S. Utility application Ser. No. 13/835,619, filed Mar. 15, 2013 and U.S. Provisional Application Ser. No. 61/737,761, filed Dec. 15, 2012, the contents of which are hereby incorporated by references.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure relates to antimicrobial compounds, in particular to antibacterial compounds and antifungal compounds. In certain embodiments, the antimicrobial compounds are effective against pathogens of hospital-acquired infections. In certain embodiments, the antimicrobial compounds are effective against pathogens that are resistant to antibiotics.

2. Description of the Background

Widespread use of antibiotics in recent decades has led to proliferation of pathogens having multiple drug resistance, often encoded by transmissible plasmids, and therefore capable of spreading rapidly between species. Many previously useful antibiotics are no longer effective against infectious organisms isolated from human and animal subjects. The specter of epidemic forms of bacterial diseases such as tuberculosis and fungal diseases, which are refractory to known antibiotic agents, may be realized in the near future. Development of novel antimicrobial compounds is a continuing urgent public health need.

Antibiotic-resistant strains of pathogenic microbes are a particular concern in cases of nosocomial infections. A nosocomial infection, also known as a hospital-acquired infection or HAI, is an infection whose development is favored by a hospital environment, such as one acquired by a patient during a hospital visit or one developing among hospital staff. Such infections include fungal and bacterial infections and are aggravated by the reduced resistance of individual patients, in particular, immune-compromised patients. The 10 most common pathogens (accounting for 84% of any reported HAIs in the U.S. in 2006-2007) were coagulase-negative staphylococci (15%), Staphylococcus aureus (15%), Enterococcus species (12%), Candida species (11%), Escherichia coli (10%), Pseudomonas aeruginosa (8%), Klebsiella pneumoniae (6%), Enterobacter species (5%), Acinetobacter baumannii (3%), and Klebsiella oxytoca (2%). The pooled mean proportion of pathogenic isolates resistant to antimicrobial agents varied significantly across types of HAI for some pathogen-antimicrobial combinations. As many as 16% of all HAIs were associated with the following multidrug-resistant pathogens: methicillin-resistant S. aureus (MRSA) (8% of HAIs), vancomycin-resistant Enterococcus faecium (4%), carbapenem-resistant P. aeruginosa (2%), extended-spectrum cephalosporin-resistant K. pneumoniae (1%), extended-spectrum cephalosporin-resistant E. coli (0.5%), and carbapenem-resistant A. baumannii, K. pneumoniae, K. oxytoca, and E. coli (0.5%). Hidron, A., et al., Antimicrobial-Resistant Pathogens Associated With Healthcare-Associated Infections: Annual Summary of Data Reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007, Infect. Control Hosp. Epidemiol. 2008; 29:996-1011, abstract.

SUMMARY OF THE INVENTION

The present disclosure provides antimicrobial compounds, compositions comprising such antimicrobial compounds, and methods of their use, in particular, antibacterial compounds and antifungal compounds. In certain aspects, the antimicrobial compounds are effective against pathogens of hospital-acquired infections. In certain aspects, the antimicrobial compounds are effective against pathogens that are resistant to antibiotics. The antimicrobial compounds can be used in antibacterial compositions, antifungal compositions, antiseptic compositions and disinfectant compositions. The antimicrobial compounds can be used as adjuncts in antibacterial compositions and antifungal compositions.

In certain embodiments, the antimicrobial compound inhibits growth of a bacteria (e.g., cutaneous, mucosal, or enteric bacteria), fungus, or virus. In preferred embodiments, with respect to bacteria, the antimicrobial compound inhibits growth of a cell selected from the genera consisting of Acinetobacter, Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Corynebacterium, Haemophilus, Proteus, Pseudomonas, Serratia, Staphylococcus, and Streptococcus. In preferred embodiments, with respect to fungi, the antimicrobial compound inhibits growth of a cell selected from the genera consisting of Aspergillus, and Candida.

The compound of Formula I is provided

where n is an integer from 6-12 inclusive; R¹, and R², are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, CH₃, CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂; R³, and R⁴, are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, C₆-C₁₀ aryl or C₆-C₁₀ heteroaryl, CH₃, CH(CH₃)₂, CH₂CHOH, CH₂CH(CH₃)₂, and (CH₂)₂N(CH₂CH₃)₂; R⁵, and R⁶, are the same or different and are selected independently from the group consisting of H and CH₃, and pharmaceutically acceptable salts thereof. In certain embodiments, the compound of Formula I is configured wherein R¹ is H and R² is selected from the group consisting of CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂. In certain embodiments, the compound of Formula I is configured wherein R¹ is CH₃ and R² is CH₃. In other embodiments, the compound Formula I is configured wherein R³ is H and R⁴ is selected from the group consisting of CH(CH₃)₂, CH₂CHOH, CH₂CH(CH₃)₂, and (CH₂)₂N(CH₂CH₃)₂. Typically, the salt of the compound is selected from the group consisting of hydrochloride, phosphate, maleate, 2-hydroxypropane-1,2,3-tricarboxylate, sulfonate, methane sulfonate, ethane sulfonate, 2-hydroxyethane sulfonate, benzene sulfonate, 4-methyl-benzene sulfonate, and heminaphthalene-1,5-disulfonate.

In preferred embodiments, the compound of Formula I is provided

where n is an integer from 6-12 inclusive; R¹, and R², are the same or different and are selected independently from the group consisting of H, CH₃, CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂; R³, and R⁴, are the same or different and are selected independently from the group consisting of H, CH₃, CH(CH₃)₂, CH₂CHOH, CH₂CH(CH₃)₂, and (CH₂)₂N(CH₂CH₃)₂; R⁵, and R⁶, are the same or different and are selected independently from the group consisting of H and CH₃, and pharmaceutically acceptable salts thereof. In certain embodiments, the compound of Formula I is configured wherein R¹ is H and R² is selected from the group consisting of CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂. In certain embodiments, the compound of Formula I is configured wherein R¹ is CH₃ and R² is CH₃. In other embodiments, the compound Formula I is configured wherein R³ is H and R⁴ is selected from the group consisting of CH(CH₃)₂, CH₂CHOH, CH₂CH(CH₃)₂, and (CH₂)₂N(CH₂CH₃)₂. Typically, the salt of the compound is selected from the group consisting of hydrochloride, phosphate, maleate, 2-hydroxypropane-1,2,3-tricarboxylate, sulfonate, methane sulfonate, ethane sulfonate, 2-hydroxyethane sulfonate, benzene sulfonate, 4-methyl-benzene sulfonate, and heminaphthalene-1,5-disulfonate.

Exemplary compounds include dodecyl 2-(dimethylamino) propanoate 4-methylbenzenesulfonate, dodecyl 2-(dimethylamino) propanoate sulfate, dodecyl 2-(dimethylamino) propanoate 2-hydroxypropane-1,2,3-tricarboxylate, dodecyl 2-(dimethylamino) propanoate phosphate, dodecyl 2-(dimethylamino) propanoate benzenesulfonate, dodecyl 2-(dimethylamino) propanoate maleate, dodecyl 2-(dimethylamino) propanoate methanesulfonate, dodecyl 2-(dimethylamino) propanoate ethanesulfonate, dodecyl 2-(dimethylamino) propanoate heminaphthalene-1,5-disulfonate, dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate, dodecyl 2-(dimethylamino)-3-hydroxybutanoate hydrochloride, dodecyl 2-(dimethylamino) acetate hydrochloride, dodecyl 2-(dimethylamino)-3-methylbutanoate hydrochloride, dodecyl 2-(dimethylamino)-3-phenylpropanoate hydrochloride, dodecyl 2-(dimethylamino)-4-methylpentanoate hydrochloride, D-dodecyl 2-(dimethylamino) propanoate hydrochloride, L-dodecyl 2-(dimethylamino) propanoate hydrochloride, dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride, dodecyl 2-(methylamino) propanoate hydrochloride, dodecyl 2-(isopropylamino) propanoate hydrochloride, dodecyl 2-((2-hydroxyethyl)amino) propanoate hydrochloride, dodecyl 2-((2-(diethylamino)ethyl)amino) propanoate dihydrochloride, tridecan-2-yl 2-(dimethylamino) propanoate hydrochloride, 2-methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride, tetradecyl 2-(dimethylamino) propanoate hydrochloride, undecyl 2-(dimethylamino) propanoate hydrochloride, decyl 2-(dimethylamino) propanoate hydrochloride, tridecyl 2-(dimethylamino) propanoate hydrochloride, octyl 2-(dimethylamino) propanoate hydrochloride, and tridecan-2-yl 2-(dimethylamino)-2-methylpropanoate.

In certain embodiments, the compound is selected from the group consisting of dodecyl 2-(methylamino) propanoate hydrochloride, dodecyl 2-(isopropylamino) propanoate hydrochloride, dodecyl 2-((2-hydroxyethyl)amino) propanoate hydrochloride, and dodecyl 2-((2-(diethylamino)ethyl)amino) propanoate dihydrochloride.

In certain embodiments, the compound is selected from the group consisting of

and pharmaceutically acceptable salts thereof.

In other aspects, a method of inhibiting the growth of a microorganism is provided, comprising the steps of providing an effective amount of the compound of formula I

where n is an integer from 6-12 inclusive; R¹, and R², are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, CH₃, CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂; R³, and R⁴, are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, C₆-C₁₀ aryl or C₆-C₁₀ heteroaryl, CH₃, CH(CH₃)₂, CH₂CHOH, CH₂CH(CH₃)₂, and (CH₂)₂N(CH₂CH₃)₂; R⁵, and R⁶, are the same or different and are selected independently from the group consisting of H and CH₃, and pharmaceutically acceptable salts thereof. In certain embodiments, the compound of Formula I is configured wherein R¹ is H and R² is selected from the group consisting of CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂. Typically, the microorganism is a member of a genus selected from the group consisting of Acinetobacter, Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Corynebacterium, Haemophilus, Proteus, Pseudomonas, Serratia, Staphylococcus, Streptococcus, Aspergillus, and Candida.

In other aspects, a disinfectant composition is provided, comprising an effective amount of the compound of formula I

where n is an integer from 6-12 inclusive; R¹, and R², are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, CH₃, CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂; R³, and R⁴, are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, C₆-C₁₀ aryl or C₆-C₁₀ heteroaryl, CH₃, CH(CH₃)₂, CH₂CHOH, CH₂CH(CH₃)₂, and (CH₂)₂N(CH₂CH₃)₂; R⁵, and R⁶, are the same or different and are selected independently from the group consisting of H and CH₃, and pharmaceutically acceptable salts thereof. In certain embodiments, the compound of Formula I is configured wherein R¹ is H and R² is selected from the group consisting of CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂. and contacting the microorganism with the compound. Also provided is a method of sanitizing a surface comprising treating the surface with such a disinfectant composition.

In other aspects, a surface having a coating of an antimicrobially effective amount of the compound of formula I

where n is an integer from 6-12 inclusive; R¹, and R², are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, CH₃, CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂; R³, and R⁴, are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, C₆-C₁₀ aryl or C₆-C₁₀ heteroaryl, CH₃, CH(CH₃)₂, CH₂CHOH, CH₂CH(CH₃)₂, and (CH₂)₂N(CH₂CH₃)₂; R⁵, and R⁶, are the same or different and are selected independently from the group consisting of H and CH₃, and pharmaceutically acceptable salts thereof. In certain embodiments, the compound of Formula I is configured wherein R¹ is H and R² is selected from the group consisting of CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂. The surface can be that of a bandage or a surgical instrument.

The above described and other features are exemplified by the following figures and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages will be apparent from the following more particular description of exemplary embodiments of the disclosure, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the disclosure.

FIG. 1A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate hydrochloride (Nex-05).

FIG. 1B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate hydrochloride.

FIG. 1C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate hydrochloride showing a peak area of 100%. Methods: column: Dionex, Acclaim Surfactant (4.6×250 mm, 5 um); mobile phase: A: 50 mM ammonium bicarbonate (pH—7.0)/B: acetonitrile; injection volume 10 μL, column temperature, 25° C., flow rate 1.0 mL/min, isocratic A:B (30:70).

FIG. 2A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate p-toluene sulfonate salt (Nex-01).

FIG. 2B is a LCMS spectrum: 286 (M⁺+1) of d dodecyl 2-(dimethylamino) propanoate p-toluene sulfonate salt.

FIG. 2C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate p-toluene sulfonate salt showing a peak area of 80.48%. Methods as in FIG. 1C.

FIG. 3A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate sulfonic salt (Nex-03).

FIG. 3B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate sulfonic salt.

FIG. 3C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate sulfonic salt showing a peak area of 98.98%. Methods as in FIG. 1C.

FIG. 4A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate citrate salt (Nex-07).

FIG. 4B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate citrate salt.

FIG. 4C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate citrate salt showing a peak area of 88%. Methods as in FIG. 1C.

FIG. 4D is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate citrate salt showing a peak area of 88%. Methods: column: Zorbax SB Phenyl (150×4.6 mm, 3.5 μm); mobile phase: A: 50 mM ammonium bicarbonate (pH—7.0)/B: acetonitrile; injection volume 10 μL, column temperature, 25° C., flow rate 1.4 mL/min, isocratic A:B (30:70).

FIG. 5A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate phosphorate salt (Nex-15).

FIG. 5B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate phosphorate salt.

FIG. 5C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate phosphorate salt showing a peak area of 95.8%. Methods as in FIG. 1C.

FIG. 6A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt (Nex-16).

FIG. 6B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt.

FIG. 6C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt showing a peak area of 87.5%. Methods as in FIG. 1C.

FIG. 6D is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt showing a peak area of 87.3%. Methods as in FIG. 4D.

FIG. 7A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate maleate salt (Nex-20).

FIG. 7B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate maleate salt.

FIG. 7C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate maleate salt showing a peak area of 87.8%. Methods as in FIG. 1C.

FIG. 7D is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate maleate salt showing a peak area of 85.9%. Methods as in FIG. 4D.

FIG. 8A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate methane sulfonate salt (Nex-22).

FIG. 8B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate methane sulfonate salt.

FIG. 8C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate methane sulfonate salt showing a peak area of 100.0%. Methods as in FIG. 1C.

FIG. 8D is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate methane sulfonate salt showing a peak area of 100.0%. Methods as in FIG. 4D.

FIG. 9A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate ethane sulfonate salt (Nex-30).

FIG. 9B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate ethane sulfonate salt.

FIG. 9C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate ethane sulfonate salt showing a peak area of 99.5%. Methods as in FIG. 1C.

FIG. 10A is a ¹H-NMR spectrum (400 MHz, CDCl₃) dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate salt (Nex-32).

FIG. 10B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate salt.

FIG. 10C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate salt showing a peak area of 87.2%. Methods as in FIG. 1C.

FIG. 11A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate salt (Nex-46).

FIG. 11B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate salt.

FIG. 11C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate salt showing a peak area of 100%. Methods as in FIG. 1C.

FIG. 12A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino)-3-hydroxybutanoate.HCl salt (Nex-51).

FIG. 12B is a LCMS spectrum: 316 (M⁺+1) of dodecyl 2-(dimethylamino)-3-hydroxybutanoate.HCl salt.

FIG. 12C is a HPLC chromatogram of dodecyl 2-(dimethylamino)-3-hydroxy-butanoate.HCl salt showing a peak area of 100%. Methods as in FIG. 4D.

FIG. 13A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino)acetate.HCl salt (Nex-52).

FIG. 13B is a LCMS spectrum: 272 (M⁺+1) of dodecyl 2-(dimethylamino)acetate.HCl salt.

FIG. 13C is a HPLC chromatogram of dodecyl 2-(dimethylamino)acetate.HCl salt showing a peak area of 99.28%. Methods as in FIG. 4D.

FIG. 14A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) 3-methylbutanoate.HCl salt (Nex-53).

FIG. 14B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 14A.

FIG. 14C is a LCMS spectrum: 314 (M⁺+1) of dodecyl 2-(dimethylamino) 3-methylbutanoate.HCl salt.

FIG. 14D is a HPLC chromatogram of dodecyl 2-(dimethylamino) 3-methylbutanoate.HCl salt showing a peak area of 99.98%. Methods as in FIG. 4D.

FIG. 15A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) 3-phenyl propanoate.HCl salt (Nex-54).

FIG. 15B is a LCMS spectrum: 314 (M⁺+1) of dodecyl 2-(dimethylamino) 3-phenyl propanoate.HCl salt.

FIG. 15C is a HPLC chromatogram of dodecyl 2-(dimethylamino) 3-phenyl propanoate.HCl salt showing a peak area of 99.98%. Methods as in FIG. 4D

FIG. 16A is a ¹H-NMR spectrum of dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl salt (Nex-55).

FIG. 16B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 16A.

FIG. 16C is a LCMS spectrum: 328 (M⁺+1) of dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl salt.

FIG. 16D is a HPLC chromatogram of dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl salt showing a peak area of 99.92%. Methods as in FIG. 4D.

FIG. 17A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of D-dodecyl 2-(dimethylamino) propanoate hydrochloride salt (Nex-56).

FIG. 17B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 17A.

FIG. 17C is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 17A.

FIG. 17D is a LCMS spectrum: 286.26 (M⁺+1) of D-dodecyl 2-(dimethylamino) propanoate hydrochloride salt.

FIG. 17E is a HPLC chromatogram of D-dodecyl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.92%. Methods as in FIG. 4D.

FIG. 17F is a HPLC chromatogram of the DL-dodecyl 2-(dimethylamino) propanoate hydrochloride salt racemate showing the separate peaks of the two stereoisomers. Methods: column: Chiralpak AD-3 (250×4.6 mm, 3 μm); mobile phase: 0.1% diethyl amine in Methanol (100:0.1); injection volume 10 μL, column temperature, 15° C., flow rate 1.0 mL/min; detection 235 nm 4 nm.

FIG. 17G is a HPLC chromatogram of D-dodecyl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.92%. Methods as in FIG. 17F, except that the detection was 235 nm 8 nm.

FIG. 18A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt (Nex-57).

FIG. 18B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 18A.

FIG. 18C is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 18A.

FIG. 18D is a LCMS spectrum: 286.28 (M⁺+1) of L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt.

FIG. 18E is a HPLC chromatogram of L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.85%. Methods as in FIG. 4D.

FIG. 18F is a HPLC chromatogram of L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.5%. Methods as in FIG. 17F.

FIG. 19A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride salt (Nex-58).

FIG. 19B is a LCMS spectrum: 300 (M⁺+1) of dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride salt.

FIG. 19C is a HPLC chromatogram of dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride salt showing a peak area of 98.07%. Methods as in FIG. 4D.

FIG. 20A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(methylamino) propanoate hydrochloride salt (Nex-59).

FIG. 20B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 20A.

FIG. 20C is a LCMS spectrum: 272.3 (M⁺+1) of dodecyl 2-(methylamino)-2-methylpropanoate hydrochloride salt.

FIG. 20D is a HPLC chromatogram of dodecyl 2-(methylamino) propanoate hydrochloride salt showing a peak area of 98.45%. Methods as in FIG. 4D.

FIG. 21A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(isopropyl amino) propanoate hydrochloride salt (Nex-60)

FIG. 21B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 21A.

FIG. 21C is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 21A

FIG. 21D is a LCMS spectrum: 300.31 (M⁺+1) of dodecyl 2-(isopropyl amino) propanoate hydrochloride salt.

FIG. 21E is a HPLC chromatogram of dodecyl 2-(methylamino) propanoate hydrochloride salt showing a peak area of 98.6%. Methods as in FIG. 4D.

FIG. 22A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-((2-hydroxyethyl)amino) propanoate hydrochloride salt (Nex-61).

FIG. 22B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 22A.

FIG. 22C is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 22A.

FIG. 22D is a LCMS spectrum: 302.47 (M⁺+1) of dodecyl 2-(isopropyl amino) propanoate hydrochloride salt.

FIG. 22E is a HPLC chromatogram of dodecyl 2-(methylamino) propanoate hydrochloride salt showing a peak area of 93.9%. Methods as in FIG. 4D.

FIG. 23A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-((2-(diethyl amino) ethyl)amino) propanoate hydrochloride salt (Nex-62).

FIG. 23B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 23A.

FIG. 23C is a ¹H-NMR spectrum (400 MHz, DMSO-d₆); compare to FIG. 23A

FIG. 23D is a LCMS spectrum: 357.59 (M⁺+1) of dodecyl 2-((2-(diethyl amino) ethyl)amino) propanoate hydrochloride salt.

FIG. 24A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of tridecan-2-yl 2-(dimethylamino) propanoate hydrochloride salt (Nex-64).

FIG. 24B is a LCMS spectrum: 357.59 (M⁺+1) of tridecan-2-yl 2-(dimethylamino) propanoate hydrochloride salt.

FIG. 24C is a HPLC chromatogram of tridecan-2-yl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.62%. Methods as in FIG. 4D.

FIG. 25A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of 2-methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride salt (Nex-65).

FIG. 25B is a LCMS spectrum: 314 (M⁺+1) of 2-methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride salt.

FIG. 25C is a HPLC chromatogram of 2-methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 95.7%. Methods as in FIG. 4D.

FIG. 26A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of tetradecyl-2-N,N-dimethylaminopropionate hydrochloride (Nex-66).

FIG. 26B is a ¹³C NMR spectrum (400 MHz, CDCl₃); compare to FIG. 26A.

FIG. 26C is a LCMS spectrum: 314 (M⁺+1) of tetradecyl-2-N,N-dimethylaminopropionate hydrochloride salt.

FIG. 26D is a HPLC chromatogram of tetradecyl-2-N,N-dimethyl aminopropionate hydrochloride salt showing a peak area of 99.7%. Methods as in FIG. 4D.

FIG. 27A is a ¹H-NMR spectrum (400 MHz, DMSO-d6) of undecyl-2-N,N-dimethylaminopropionate hydrochloride (Nex-67).

FIG. 27B is a ¹³C NMR spectrum (400 MHz, CDCl₃); compare to FIG. 27A.

FIG. 27C is a LCMS spectrum: 272 (M⁺+1) of undecyl-2-N,N-dimethylamino-propionate hydrochloride salt.

FIG. 27D is a HPLC chromatogram of undecyl-2-N,N-dimethylaminopropionate hydrochloride salt showing a peak area of 99.6%. Methods as in FIG. 4D.

FIG. 28A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of decyl-2-N,N-dimethyl-aminopropionate hydrochloride (Nex-68).

FIG. 28B is a ¹³C NMR spectrum (400 MHz, CDCl₃); compare to FIG. 28A.

FIG. 28C is a LCMS spectrum: 258 (M⁺+1) of decyl-2-N,N-dimethylamino-propionate hydrochloride salt.

FIG. 28D is a HPLC chromatogram of decyl-2-N,N-dimethylaminopropionate hydrochloride salt showing a peak area of 99.18%. Methods as in FIG. 4D.

FIG. 29A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of tridecyl-2-N,N-dimethyl-aminopropionate hydrochloride (Nex-69).

FIG. 29B is a ¹³C NMR spectrum (400 MHz, CDCl₃); compare to FIG. 29A.

FIG. 29C is a LCMS spectrum: 258 (M⁺+1) of tridecyl-2-N,N-dimethylamino-propionate hydrochloride salt.

FIG. 29D is a HPLC chromatogram of tridecyl-2-N,N-dimethylaminopropionate hydrochloride salt showing a peak area of 99.18%. Methods as in FIG. 4D.

FIG. 30A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of octyl-2-N,N-dimethyl-aminopropionate hydrochloride (Nex-70).

FIG. 30B is a ¹³C NMR spectrum (400 MHz, CDCl₃); compare to FIG. 30A.

FIG. 30C is a LCMS spectrum: 258 (M⁺+1) of octyl-2-N,N-dimethylamino-propionate hydrochloride salt.

FIG. 30D is a HPLC chromatogram of octyl-2-N,N-dimethylaminopropionate hydrochloride salt showing a peak area of 99.18%. Methods as in FIG. 4D.

FIG. 31A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of tridecan-2-yl 2-(dimethylamino) 2-methyl propanoate hydrochloride (Nex-71).

FIG. 31B is a ¹H-NMR spectrum (400 MHz, CDCl₃); compare to FIG. 31A.

FIG. 31C is a LCMS spectrum: 314 (M⁺+1) of tridecan-2-yl 2-(dimethylamino) 2-methyl propanoate hydrochloride salt.

FIG. 31D is a HPLC chromatogram of tridecan-2-yl 2-(dimethylamino) 2-methyl propanoate hydrochloride salt showing a peak area of 94.48%. Methods as in FIG. 4D.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein and in the appended claims, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to bacteria includes a plurality of bacteria species. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

Where the compounds according to the invention have at least one asymmetric center, they may accordingly exist as enantiomers. Where the compounds according to the invention possess two or more asymmetric centers, they may additionally exist as diastereoisomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention. The examples of the use of stereroisomeric compounds in the practice of the present invention disclosed herein are illustrative examples, and are not limiting.

Practice of the embodiments of the invention also involves pharmaceutical compositions comprising one or more compounds of this invention in association with a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. It is also envisioned that the compounds of the present invention may be incorporated into transdermal patches designed to deliver the appropriate amount of the drug in a continuous fashion. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture for a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be easily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Typical unit dosage forms contain from 1 to 100 mg, for example, 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

The liquid forms in which the compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.

Additional ingredients such as coloring agents, anti-microbial preservatives, emulsifiers, perfumes, active ingredient stabilizers, and the like may be included in the compositions as long as the resulting composition retains desirable properties, as described above. When present, preservatives are usually added in amounts of about 0.05 to about 0.30%. Suitable preservatives include methylparabens (methyl PABA), propylparabens (propyl PABA) and butylhydroxy toluene (BHT). Suitable perfumes and fragrances are known in the art; a suitable fragrance is up to about 5 percent myrtenol, preferably about 2 percent myrtenol, based on the total weight of the composition.

The term “antimicrobial” refers to an ability to prevent, resist, kill, or inhibit the growth of microorganisms (including, without limitation, viruses, bacteria, yeast, fungi, protozoa, etc.), or to attenuate the severity of a microbial infection. The antimicrobial compounds of the present invention are compounds that may be used in the treatment of disease and infection or preservation of an uninfected surface.

The term “active antimicrobial agent” as used herein, refers to compounds with known activity for the treatment of disease caused by microbes, and in particular agents that are effective in sublingual, intraocular, intraaural, and particularly topical, application.

The term “active pharmaceutical ingredient” means any substance or mixture of substances intended to be used in the manufacture of a drug (medicinal) product and that when used in the production of a drug becomes an active ingredient of the drug product. Such substances are intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease or to effect the structure and function of the body.

The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease (or infection) and/or adverse effect attributable to the disease (or infection). The terms “treatment”, “treating” and the like as used herein includes:

(a) preventing a microbial disease and/or infection from occurring in a subject who may be predisposed to but has not yet been diagnosed as having it;

(b) inhibiting the progress or transmission of a microbial disease and/or infection, i.e., arresting its development or maintenance; or

(c) relieving a bacterial disease (i.e., causing regression and/or amelioration of the disease) and/or infection.

Bactericidal and/or bacteriostatic activity of the disclosed compositions including compounds of the invention may be measured using any number of methods available to those skilled in the art. One example of such a method is measurement of antibacterial activity through use of a MIC (minimal inhibitory concentration) test that is recognized to be predictive of in vivo efficacy for the treatment of a bacterial infection with antibiotics. In preferred embodiments, the disclosed compositions display antibacterial activity in this test, even without pretreatment of the bacteria to permeabilize the membrane.

In preferred embodiments, the present invention provides methods of inhibiting the growth of microorganisms by contacting the microorganisms with compositions of the invention in which the active agent is disclosed antimicrobial compound. These methods are effective against infections in vivo, and particularly topical infections. This is demonstrated by test data showing the minimum inhibitory concentrations (MIC) and time kill studies of compositions against various pathogenic organisms cultured in vitro under standard conditions. These in vitro tests strongly correlate with in vivo activity, as is evidenced by the widespread use of the MIC determinations to predict utility of antimicrobial compositions in treatment of infection in animals, including humans.

Compositions of the invention may be provided as topical disinfectants for sterilization of surfaces such as countertops, surgical instruments, bandages, patches, medical devices, and skin; as pharmaceutical compositions, including by way of example creams, lotions, ointments, gels, sprays, or solutions for external application to skin and mucosal surfaces, including the cornea, dermal cuts and abrasions, burns, and sites of bacterial or fungal infection; as pharmaceutical compositions, including by way of example creams, lotions, ointments, emulsions, liposome dispersions, gaseous suspension of fine solid or liquid particles, or formulations, suppositories, or solutions, for administration to internal mucosal surfaces such as the oral cavity or vagina to inhibit the growth of bacteria or fungi, including yeasts; and as pharmaceutical compositions such as creams, gels, or ointments for coating indwelling catheters and similar implants which are susceptible to harboring bacterial or fungal infection.

Particular formulations may be manufactured according to methods well known in the art. Formulations are given in, for example, Remington's The Science and Practice of Pharmacy and similar reference works.

In certain embodiments, the disclosed antimicrobial compounds are useful as stabilizing and/or preservative compounds in topical antibiotic compositions, both prescription (e.g., benzomycin creams) and over-the-counter (e.g., anti-acne medications containing salicylic acid, benzoyl peroxide and the like.) When used in the therapeutic treatment of disease, an appropriate dosage of a composition containing the disclosed antimicrobial compounds of the invention and an active ingredient may be determined by any of several well established methodologies. For instance, animal studies are commonly used to determine the maximal tolerable dose, or MTD, of bioactive agent per kilogram weight. In general, at least one of the animal species tested is mammalian. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human. Additionally, therapeutic dosages may also be altered depending upon factors such as the severity of infection, and the size or species of the host.

Where the therapeutic use of the presently described antimicrobial compositions is contemplated, the compositions are preferably administered in a pharmaceutically acceptable topical carrier. Besides the pharmaceutically acceptable topical carrier, the composition of the invention can also comprise additives, such as stabilizers, excipients, buffers and preservatives. Typically, but not necessarily, the preferred formulation for a given antimicrobial composition is dependant on the location in a host where a given infectious organism would be expected to initially invade, or where a given infectious organism would be expected to colonize or concentrate. For example, topical infections are preferably treated or prevented by formulations designed for application to specific body surfaces, e.g., skin, mucous membranes, etc. In such an embodiment, the composition containing the antimicrobial compound is formulated in a water, ethanol, and propylene glycol base for topical administration. Alternatively, where the targeted pathogen colonizes nasal passages, compositions suitable for intranasal administration can be formulated. For such a targeted pathogen colony, a buccal spray may be a preferred method of delivery.

Preferably, animal subjects that may be treated using the compositions of the present invention include, but are not limited to, invertebrates, vertebrates, birds, mammals such as pigs, goats, sheep, cows, dogs, cats, and particularly humans. The presently described compositions are also contemplated to be effective in combating bacterial contamination of laboratory cultures, consumables (food or beverage preparations), medical devices, hospital apparatus, or industrial processes.

Given that bacterial and fungal infections are particularly problematic in immuno-compromised individuals, such as patients suffering from acquired immunodeficiency disease syndrome (AIDS), HIV-infected individuals, patients undergoing chemotherapy or radiation therapy, or bone marrow transplantation, etc., an additional embodiment of the presently described invention is the use of the presently described antimicrobial compounds as prophylactic agents to prevent and/or treat infection in immuno-compromised patients.

Examples of bacterial organisms against which the methods and compositions of the invention are effective include gram positive bacteria, gram negative bacteria, and acid fast bacteria, and particularly, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Escherichia coli.

A range of fungi or moulds, called dermatophytes, cause fungal infections of the skin. These fungi are parasites on the skin and cause different symptoms in different parts of the body. They are very infectious and are passed from person to person. Although typically these infections are topical, in certain patients (e.g., immunosuppressed patients) they may occur systemically or internally. Vaginal yeast infections are generally caused by Candida albicans, which, along with a few types of bacteria, are normally present in relatively small numbers in the vaginal area. Sometimes the yeast multiply rapidly and take over, causing candidiasis or monilia. This is often due to a change in the vaginal environment, injury, sexual transmission, HIV infection, etc. Common environmental disruptions that favor yeast include increased pH, increased heat and moisture, allergic reactions, elevated sugar levels, hormonal fluxes, and reductions in the populations of bacteria that are normally present.

In further embodiments, the disclosed antimicrobial compounds can also be used as adjuncts in conjunction with conventional antimicrobial agents in compositions of the present invention. The added activity of the active ingredients may provide for a more efficacious composition, and can provide multiple mechanisms by which the microbes are targeted.

The structural formulas and characteristics of the antimicrobial compounds are summarized in Table 1, below. Further details of the methods of making and methods of use of these antimicrobial compounds are provided in working Examples 1-35, below.

TABLE 1 Base Salt Code Name Structure MW MW  g  Appear. Sol. pH  1 Nex-01 Dodecyl 2- (dimethylamino) propanoate 4-methyl- benzenesulfonate

285 457 100 White powder  35% 4.25  2 Nex-03 Dodecyl 2- (dimethylamino) propanoate sulfate

285 383 100 White powder  37% 1.60  3 Nex-05 Dodecyl 2- (dimethylamino) propanoate hydrochloride

285 321 100 White powder  31% 2.87  4 Nex-07 Dodecyl 2- (dimethylamino) propanoate 2- hydroxypropane- 1,2,3- tricarboxylate

285 477 100 White powder  36% 3.24  5 Nex-15 Dodecyl 2- (dimethylamino) propanoate phosphate

285 383 100 White paste  33% 2.01  6 Nex-16 Dodecyl 2- (dimethylamino) propanoate benzenesulfonate

285 443 100 White powder  33% 3.40  7 Nex-20 Dodecyl 2- (dimethylamino) propanoate maleate

285 401 100 White powder  31% 3.39  8 Nex-22 Dodecyl 2- (dimethylamino) propanoate methanesulfonate

285 381 100 White powder  35% 3.25  9 Nex-30 Dodecyl 2- (dimethylamino) propanoate ethanesulfonate

285 395 100 White powder  33% 3.79 10 Nex-32 Dodecyl 2- (dimethylamino) propanoate heminaphthalene- 1,5-disulfonate

285 859 100 White powder  34% 3.87 11 Nex-46 Dodecyl 2- (dimethylamino) propanoate 2- hydroxyethanesulfonate

285 411 100 Waxy solid  33% 2.95 12 Nex-51 Dodecyl 2- (dimethylamino)-3- hydroxybutanoate hydrochloride

315 352  22 White jelly thick liquid  98% 0.56 13 Nex-52 Dodecyl 2- (dimethylamino) acetate hydrochloride

271 308  26 White solid  49% 3.30 14 Nex-53 Dodecyl 2- (dimethylamino)-3- methylbutanoate hydrochloride

314 350  25 White solid  44% 1.69 15 Nex-54 Dodecyl 2- (dimethylamino)-3- phenylpropanoate hydrochloride

362 398  36 White powder  58% 2.16 16 Nex-55 Dodecyl 2- (dimethylamino)-4- methylpentanoate hydrochloride

328 364  25 White solid  50% 1.84 17 Nex-56 D-Dodecyl 2- (dimethylamino) propanoate hydrochloride

285 321  25 White powder  50% 1.70 18 Nex-57 L-Dodecyl 2- (dimethylamino) propanoate hydrochloride

285 321  25 White solid  53% 1.57 19 Nex-58 Dodecyl 2- (dimethylamino)-2- methylpropanoate hydrochloride

299 336  31 White solid  51% 2.27 20 Nex-59 Dodecyl 2- (methylamino) propanoate hydrochloride

271 308  18 White powder  33% 2.17 21 Nex-60 Dodecyl 2- (isopropylamino) propanoate hydrochloride

299 336  20 White solid  46% 2.04 22 Nex-61 Dodecyl 2-((2- hydroxyethyl)amino) propanoate hydrochloride

301 338  21 White solid  40% 1.50 23 Nex-62 Dodecyl 2-((2- (diethylamino)ethyl) amino) propanoate dihydrochloride

357 430  25 White solid  48% 0.73 24 Nex-64 Tridecan-2-yl 2- (dimethylamino) propanoate hydrochloride

299 336  25 White powder  44% 1.95 25 Nex-65 2-Methyltridecan- 2-yl 2- (dimethylamino) propanoate hydrochloride

314 350  17 White powder  51% 1.66 26 Nex-66 Tetradecyl 2- (dimethylamino) propanoate hydrochloride

314 350  11 White powder  48% 2.35 27 Nex-67 Undecyl 2- (dimethylamino) propanoate hydrochloride

271 308  15 Waxy solid  81% 3.77 28 Nex-68 Decyl 2- (dimethylamino) propanoate hydrochloride

257 294  14 White powder  97% 0.87 29 Nex-69 Tridecyl 2- (dimethylamino) propanoate hydrochloride

299 336  10 White powder  51% 1.64 30 Nex-70 Octyl 2- (dimethylamino) propanoate hydrochloride

229 266  18 Waxy solid 134% 0.88 31 Nex-71 Tridecan-2-yl 2- (dimethylamino)-2- methylpropanoate

314 N/A  13 Waxy solid N/A N/A

WORKING EXAMPLES

The following non-limiting examples further illustrate the various embodiments described herein. Example 1 provides a method of synthesizing dodecyl 2-(dimethylamino) propanoate (DDAIP) and dodecyl 2-(dimethylamino) propanoate hydrochloride salt. Examples 2-12 disclose methods of synthesizing other salts of DDAIP. The salts of DDAIP that were prepared include the sulfate, the phosphate, and organic salts including the 4-methylbenzenesulfonate, the 2-hydroxypropane-1,2,3-tricarboxylate, benzenesulfonate, the maleate, the methanesulfonate, the ethanesulfonate, the heminaphthalene-1,5-disulfonate, and the 2-hydroxyethanesulfonate. Examples 13-32 describe the methods of making and the characterization of related compounds and their hydrochloride salts. In view of the teachings of Examples 2-12 and the knowledge of the skilled artisan, the production of salts disclosed herein, as well as other salts, of the antimicrobial compounds is routine.

Example 1 Synthesis of Dodecyl 2-(dimethylamino) propanoate and Dodecyl 2-(dimethylamino) propanoate Hydrochloride salt

Synthesis of Dodecyl 2-aminopropanoate (3)

To a stirred solution of DL-alanine 1 (5 g, 56.1 mmol) in toluene (100 mL) was added dodecanol 2 (9.42 g, 50.5 mmol) in one lot, followed by pTSA (11.75 g, 61.7 mmol). After addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude dodecyl 2-aminopropanoate 3 (14.4 g, yield: 100%) as a liquid.

Synthesis of Dodecyl 2-(dimethylamino) Propanoate (DDAIP) (4)

To a stirred solution of dodecyl 2-aminopropanoate 3 (5 g, 19.4 mmol) in DCM (100 mL) was added aqueous formaldehyde solution (37% w/v) (2.03 g, 67.9 mmol) in one lot at 0° C. and slowly added Na (OAc)₃BH (10.29 g, 48.5 mmol) over a period of ½ h. After addition, the temperature of the reaction mixture was slowly raised to room temperature (RT), stirred at RT for 24 h; the reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×40 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford dodecyl 2-(dimethylamino) propanoate (DDAIP) 4 (4.2 g, yield: 75.9%) as a liquid.

Synthesis of Dodecyl 2-(dimethylamino) propanoate Hydrochloride (5, Nex-05)

A stirred solution of dodecyl 2-(dimethylamino) propanoate 4 (5 g, 17.54 mmol) in ethyl acetate/hexane/MeOH (10:10:1 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×52 mL) followed by hexane (5×50 mL) to afford wet dodecyl 2-(dimethylamino) propanoate hydrochloride 5 (5.5 g) as a semi solid. The semi solid was taken in ethyl acetate/hexane (10:10 mL) and heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained solid was filtered under nitrogen and dried under vacuum to afford dodecyl 2-(dimethylamino) propanoate. HCl salt 5 (3 g, yield: 53.5%) as a white hygroscopic solid, mp: 86-92° C.

FIG. 1A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate hydrochloride (Nex-05).

FIG. 1B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate hydrochloride.

FIG. 1C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate hydrochloride showing a peak area of 100%. Methods: column: Dionex, Acclaim Surfactant (4.6×250 mm, 5 um); mobile phase: A: 50 mM ammonium bicarbonate (pH—7.0)/B: acetonitrile; injection volume 10 μL, column temperature, 25° C., flow rate 1.0 mL/min, isocratic A:B (30:70).

Example 2 Synthesis of Dodecyl 2-(dimethylamino) propanoate p-toluene sulfonate salt (Nex-01)

Synthesis of Dodecyl 2-(dimethylamino) propanoate p-toluene sulfonate salt (7, Nex-01)

A stirred solution of DDAIP base 4 (80 g, 280 mmol) in ethyl acetate (500 mL) was cooled to 0° C. then p-toluene sulfonic acid H₂O 6 (53.4 g, 280 mmol) was added in one lot. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 12 h; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum and flushed with hexane. The obtained residue was taken in n-hexane (20 mL) and stirred at RT for 2 h (No solid). The obtained sticky solid kept in a deep freezer for 12 h to afford dodecyl 2-(dimethylamino) propanoate PTSA salt (7, Nex-01) (130 g, yield: 97.4%) as a hygroscopic solid, Mp: 60-65° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.9 (t, 3H), 1.3 (m, 18H), 1.6 (d, 3H), 1.6 (q, 2H), 2.35 (s, 3H), 3 (m, 6H), 4.1 (t, 2H), 4.25 (q, 1H), 7.18 (d, 2H), 7.78 (d, 2H); LCMS: 286 (M⁺+1); HPLC: 80.48%.

FIG. 2A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate p-toluene sulfonate salt (Nex-01).

FIG. 2B is a LCMS spectrum: 286 (M⁺+1) of d dodecyl 2-(dimethylamino) propanoate p-toluene sulfonate salt.

FIG. 2C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate p-toluene sulfonate salt showing a peak area of 80.48%. Methods as in FIG. 1C.

Example 3 Synthesis of Dodecyl 2-(dimethylamino) propanoate sulfonate salt (Nex-03)

Synthesis of Dodecyl 2-(dimethylamino) propanoate sulfonate salt (9, Nex-03)

A stirred solution of DDAIP base 4 (85 g, 298 mmol) in n-hexane (500 mL) was cooled to 0° C., and then concentrated H₂SO₄ 7 (29.22 g, 298 mmol) was added drop wise. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 12 h; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum to obtain a sticky solid. The obtained sticky solid kept in deep freezer for 12 h to afford dodecyl 2-(dimethylamino) propanoate sulfonic salt (9, Nex-03) (110 g, yield: 96.4%) as a hygroscopic solid, Mp: 58-63° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.9 (t, 3H), 1.25 (m, 18H), 1.6 (d, 3H), 1.62 (q, 2H), 3.1 (s, 6H), 4.1 (q, 1H), 4.2 (t, 2H); LCMS: 286 (M⁺+1); HPLC: 98.98%.

FIG. 3A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate sulfonic salt (Nex-03).

FIG. 3B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate sulfonic salt.

FIG. 3C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate sulfonic salt showing a peak area of 98.98%. Methods as in FIG. 1C.

Example 4 Synthesis of Dodecyl 2-(dimethylamino) propanoate hydrochloride Salt (Nex-05)

Synthesis of Dodecyl 2-(dimethylamino) propanoate HCl Salt (5, Nex-05)

A stirred solution of DDAIP base 4 (100 g, 350 mmol) in hexane (500 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 2 h, and the reaction mixture was monitored by TLC. The obtained solid was filtered under vacuum, the obtained semi solid was taken in ethyl acetate/hexane (250/250 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained solid was filtered under nitrogen and dried under vacuum to afford dodecyl 2-(dimethylamino) propanoate HCl salt (5, Nex-05) (92 g, yield: 81.56%) as a white hygroscopic solid, mp: 86-92° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.85 (t, 3H), 1.2-1.4 (m, 18H), 1.65 (q, 2H), 1.75 (d, 3H), 2.9 (s, 6H), 3.95 (q, 1H), 4.2 (t, 2H); LCMS: 286 (M⁺+1); HPLC: 100%.

Example 5 Synthesis of Dodecyl 2-(dimethylamino) propanoate citrate Salt (Nex-07)

Synthesis of Dodecyl 2-(dimethylamino) propanoate citrate salt (11, Nex-07)

A stirred solution of DDAIP base 4 (75 g, 263 mmol) in methanol (600 mL) was cooled to 0° C. then citric acid 8 (50.4 g, 0.263) was added in one lot. After addition, the temperature of the reaction mixture was slowly raised to RT, stirred at RT for 12 h, and the reaction mixture was monitored by TLC. The solvent was concentrated under vacuum. The residue was diluted with n-hexane (100 mL) which was not miscible with the product. Some seeding material was prepared (scratching the crude in a glass vial), and seeded to the crude to afford dodecyl 2-(dimethylamino) propanoate citrate salt (11, Nex-07) (120 g, yield: 95.6%) as a white solid, mp: 62-67° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.85 (t, 3H), 1.3 (m, 18H), 1.5 (d, 3H), 1.7 (q, 2H), 2.7 (m, 2H), 2.9 (s, 6H), 4.1 (q, 1H), 4.2 (t, 2H); LCMS: 286 (M⁺+1); HPLC: 88%.

FIG. 4A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate citrate salt (Nex-07).

FIG. 4B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate citrate salt.

FIG. 4C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate citrate salt showing a peak area of 88%. Methods as in FIG. 1C.

FIG. 4D is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate citrate salt showing a peak area of 88%. Methods: column: Zorbax SB Phenyl (150×4.6 mm, 3.5 μm); mobile phase: A: 50 mM ammonium bicarbonate (pH—7.0)/B: acetonitrile; injection volume 10 μL, column temperature, 25° C., flow rate 1.4 mL/min, isocratic A:B (30:70).

Example 6 Synthesis of Dodecyl 2-(dimethylamino) propanoate phosphorate Salt (Nex-15)

Synthesis of Dodecyl 2-(dimethylamino) propanoate phosphorate Salt (Nex-15)

A stirred solution of DDAIP base 4 (100 g, 350 mmol) in ethyl acetate (500 mL) was cooled to 0° C. and then phosphoric acid 12 (34.38 g, 350 mmol) was added in one lot. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 12 h; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained sticky solid was taken in n-hexane (200 mL) and stirred at RT for 2 h to afford dodecyl 2-(dimethylamino) propanoate phosphorate salt (13, Nex-15) (125 g, 93%) as a sticky solid. ¹H-NMR (400 MHz, CDCl₃): δ 0.85 (t, 3H), 1.2-1.4 (m, 18H), 1.55 (d, 3H), 1.7 (q, 2H), 2.85 (s, 6H), 4.1 (q, 1H), 4.2 (t, 2H); LCMS: 286 (M⁺+1); HPLC: 95.8%.

FIG. 5A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate phosphorate salt (Nex-15).

FIG. 5B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate phosphorate salt.

FIG. 5C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate phosphorate salt showing a peak area of 95.8%. Methods as in FIG. 1C.

Example 7 Synthesis of Dodecyl 2-(dimethylamino) propanoate benzene sulfonate Salt (Nex-16)

Synthesis of Dodecyl 2-(dimethylamino) propanoate Benzene sulfonate salt (15, Nex-16)

A stirred solution of DDAIP base 4 (75 g, 263 mmol) in ethyl acetate (500 mL) was cooled to 0° C., then benzene sulfonic acid 14 (41.55 g, 263 mmol) was added in one lot. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 24 h; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The residue washed with hexane (2×30 mL); after workup a sticky solid was observed. The obtained sticky solid was kept in deep freezer for 12 h to afford dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt (15, Nex-16) (116 g, yield: 99.5%) as a solid, mp: 55-62° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.9 (t, 3H), 1.3 (m, 18H), 1.65 (d, 3H), 1.65 (q, 2H), 3 (s, 6H), 4.2 (q, 1H), 4.2 (t, 2H), 7.4 (m, 3H), 7.9 (d, 2H); LCMS: 286 (M⁺+1); HPLC: 80.48%.

FIG. 6A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt (Nex-16).

FIG. 6B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt.

FIG. 6C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt showing a peak area of 87.5%. Methods as in FIG. 1C.

FIG. 6D is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate benzene sulfonate salt showing a peak area of 87.3%. Methods as in FIG. 4D.

Example 8 Synthesis of Dodecyl 2-(dimethylamino) propanoate maleate Salt (Nex-20)

Synthesis of Dodecyl 2-(dimethylamino) propanoate maleate salt (17, Nex-20)

A stirred solution of DDAIP base 4 (80 g, 280 mmol) in methanol (600 mL) was cooled to 0° C., then maleic acid 16 (32.48 g, 280 mmol) was added in one lot. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 12 h; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum and flushed with ethyl acetate (2×100 mL). The obtained residue was taken in n-hexane (200 mL) and stirred at RT for ½ h (No solid). The reaction mixture was concentrated under vacuum. The obtained sticky solid was kept in deep freezer for 2 h to afford dodecyl 2-(dimethylamino) propanoate maleate salt (17, Nex-20) (111 g, yield: 98.6%) as a solid, mp: 65-70° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.9 (t, 3H), 1.25 (m, 18H), 1.6 (d, 3H), 1.65 (q, 2H), 2.9 (s, 6H), 4.1 (q, 1H), 4.2 (t, 2H), 6.3 (d, 2H); LCMS: 286 (M⁺+1); HPLC: 80.48%.

FIG. 7A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate maleate salt (Nex-20).

FIG. 7B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate maleate salt.

FIG. 7C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate maleate salt showing a peak area of 87.8%. Methods as in FIG. 1C.

FIG. 7D is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate maleate salt showing a peak area of 85.9%. Methods as in FIG. 4D.

Example 9 Synthesis of Dodecyl 2-(dimethylamino) propanoate methane sulfonate Salt (Nex-22)

Synthesis of Dodecyl 2-(dimethylamino) propanoate Methane sulfonate salt (19, Nex-22)

A stirred solution of DDAIP base 4 (85 g, 298 mmol) in ethyl acetate (500 mL) was cooled to 0° C., then methane sulfonic acid 18 (28.6 g, 298 mmol) was added in one lot. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 48 h; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in n-hexane (30 mL) and stirred at RT for ½ h (No solid). The reaction mixture was concentrated under vacuum and flushed with hexane (3×30 mL) to afford dodecyl 2-(dimethylamino) propanoate methane sulfonate salt (19, Nex-22) (110 g, yield: 97.34%) as a solid, mp: 66-72° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.9 (t, 3H), 1.35 (m, 18H), 1.65 (q, 2H), 1.7 (d, 3H), 2.8 (s, 3H), 3 (s, 6H), 4.15 (q, 1H), 4.2 (t, 2H); LCMS: 286 (M⁺+1); HPLC: 100%.

FIG. 8A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate methane sulfonate salt (Nex-22).

FIG. 8B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate methane sulfonate salt.

FIG. 8C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate methane sulfonate salt showing a peak area of 100.0%. Methods as in FIG. 1C.

FIG. 8D is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate methane sulfonate salt showing a peak area of 100.0%. Methods as in FIG. 4D.

Example 10 Synthesis of Dodecyl 2-(dimethylamino) propanoate ethane sulfonate Salt (Nex-30)

Synthesis of Dodecyl 2-(dimethylamino) propanoate ethane sulfonate Salt (21, Nex-30)

A stirred solution of DDAIP base 4 (85 g, 298 mmol) in ethyl acetate (600 mL) was cooled to 0° C., then ethane sulfonic acid 20 (32.79 g, 298 mmol) was added in one lot. After addition, the temperature of the reaction mixture was slowly raised to RT, stirred at RT for 12 h and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum and flushed with hexane. The obtained residue was taken in n-hexane (200 mL) and stirred at RT for 2 h (No solid). The obtained sticky solid kept in deep freezer for 12 h to afford dodecyl 2-(dimethylamino) propanoate ethane sulfonate salt (21, Nex-30) (116 g, yield: 98.4%) as a hygroscopic solid, Mp: 45-50° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.9 (t, 3H), 1.3 (m, 18H), 1.35 (t, 3H), 1.7 (d, 3H), 1.8 (q, 2H), 2.9 (q, 2H), 3 (m, 6H), 4.2 (m, 2H), 4.2 (m, 1H); LCMS: 286 (M⁺+1); HPLC: 99.47%.

FIG. 9A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate ethane sulfonate salt (Nex-30).

FIG. 9B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate ethane sulfonate salt.

FIG. 9C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate ethane sulfonate salt showing a peak area of 99.5%. Methods as in FIG. 1C.

Example 11 Synthesis of Dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate Salt (Nex-32)

Synthesis of Dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate salt (23, Nex-32)

A stirred solution of DDAIP base 4 (80 g, 280 mmol) in methanol (500 mL) was cooled to 0° C., then 1,5-napthalene disulfonic acid 22 (50.5 g, 140 mmol) was added in one lot. After addition the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 72 h; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum and flushed with ethyl acetate (2×30 mL). The obtained residue was taken in n-hexane (200 mL) and stirred at RT for ½ h (sticky solid). The reaction mixture was concentrated under vacuum and flushed with hexane (3×100 mL) to afford dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate salt (23, Nex-32) (120 g, 92.3%) as a white solid. Mp: 135-140° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.9 (t, 3H), 1.2-1.3 (m, 18H), 1.6 (d, 3H), 1.65 (q, 2H), 2.9 (s, 6H), 4.15 (t, 2H), 4.2 (q, 1H), 7.55 (t, 2H), 8.22 (d, 2H), 9.1 (d, 2H); LCMS: 286 (M⁺+1); HPLC: 80.48%.

FIG. 10A is a ¹H-NMR spectrum (400 MHz, CDCl₃) dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate salt (Nex-32).

FIG. 10B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate salt.

FIG. 10C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate 1,5-napthalene disulfonate salt showing a peak area of 87.2%. Methods as in FIG. 1C.

Example 12 Synthesis of Dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate Salt (Nex-46)

Synthesis of Dodecyl 2-(dimethylamino) 2-hydroxyethanesulfonate salt (25, Nex-46)

To a stirred solution of DDAIP base 4 (85 g, 298 mmol) in ethyl acetate (500 mL) cooled to 0° C. was added 2-hydroxyethanesulfonic acid 24 (80% pure only) (45 g, 357 mmol) in one lot. After addition, the temperature of the reaction mixture was slowly raised to room temperature and stirred at RT for 12 h; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in n-hexane (20 mL) and stirred at RT for ½ h (No solid). The obtained sticky solid was kept in deep freezer for 48 h to afford dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate salt (25, Nex-46) (125 g, yield: 96.15%) as a waxy hygroscopic solid, Mp: 58-63° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.8 (t, 3H), 1.2-1.3 (m, 18H), 1.6 (d, 3H), 2.9 (s, 6H), 3.05 (t, 2H), 3.9 (t, 2H), 4.1 (q, 2H), 4.2 (t, 2H); LCMS: 286 (M⁺+1); HPLC: 100%.

FIG. 11A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate salt (Nex-46).

FIG. 11B is a LCMS spectrum: 286 (M⁺+1) of dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate salt.

FIG. 11C is a HPLC chromatogram of dodecyl 2-(dimethylamino) propanoate 2-hydroxyethanesulfonate salt showing a peak area of 100%. Methods as in FIG. 1C.

Example 13 Synthesis of Dodecyl 2-(dimethylamino)-3-hydroxybutanoate.HCl (Nex-51)

Synthesis of Dodecyl 2-amino-3-hydroxybutanoate (27)

To a stirred solution of DL-threonine 26 (5 g, 41.9 mmol) in toluene (100 mL) was added 1-dodecanol 2 (7 g, 37.7 mmol) in one lot, followed by pTSA (8.77 g, 46.16 mmol). After addition, the temperature of the reaction mixture was slowly raised to reflux temperature and the water was separated azeotropically. The reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 27 (11 g, yield: 91%) as a liquid.

Synthesis of Dodecyl 2-(dimethylamino)-3-hydroxybutanoate (28)

To a stirred solution of 27 (11 g, 38.3 mmol) in DCM (100 mL) was added aqueous formaldehyde solution (37% w/v) (4.02 g, 134 mmol) in one lot at 0° C., and Na (OAc)₃BH (20.3 g, 95.8 mmol) was added slowly over a period of 1 h. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 12 h, and the reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×40 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum. The obtained crude product was purified by column chromatography to afford 28 (8 g, yield: 66.6%) as a liquid.

Synthesis of Dodecyl 2-(dimethylamino)-3-hydroxybutanoate.HCl (29, Nex-51)

A stirred solution of 28 (8 g, 25.3 mmol) in hexane (50 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (5×50 mL) to afford wet dodecyl 2-(dimethylamino)-3-hydroxybutanoate.HCl (29, Nex-51) (8 g) as a semi solid. This semi solid was taken in hexane (50 mL) and heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and kept for 12 h, then to 0° C. The obtained semi solid was filtered under nitrogen & dried under vacuum to afford dodecyl 2-(dimethylamino)-3-hydroxybutanoate.HCl salt (29, Nex-51) (8 g, yield: 89.8%) as a white hygroscopic semi solid. ¹H-NMR (400 MHz, CDCl₃): δ 4.3-4.1 (m, 4H), 3.1 (s, 3H), 2.9 (s, 3H), 1.7 (m, 2H), 1.4-1.1 (m, 21H), 0.9 (t, 3H) LCMS: 316 (M⁺+1); HPLC: 99.55%.

FIG. 12A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino)-3-hydroxybutanoate.HCl salt (Nex-51).

FIG. 12B is a LCMS spectrum: 316 (M⁺+1) of dodecyl 2-(dimethylamino)-3-hydroxybutanoate.HCl salt.

FIG. 12C is a HPLC chromatogram of dodecyl 2-(dimethylamino)-3-hydroxy-butanoate.HCl salt showing a peak area of 100%. Methods as in FIG. 4D.

Example 14 Synthesis of Dodecyl 2-(dimethylamino)acetate.HCl (Nex-52)

Synthesis of Dodecyl 2-aminoacetate (31)

To a stirred solution of DL-glycine 30 (20 g, 266 mmol) in toluene (200 mL) was added 1-dodecanol 2 (44.7 g, 239.9 mmol) in one lot, followed by pTSA (55.78 g, 293 mmol). After addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude dodecyl 2-aminoacetate 31 (40 g, yield: 61.7%) as a liquid.

Synthesis of Dodecyl 2-(dimethylamino) acetate (32)

To a stirred solution of 31 (40 g, 164 mmol) in DCM (500 mL) was added aqueous formaldehyde solution (37% w/v) (17.2 g, 576 mmol) in one lot at 0° C. Na(OAc)₃BH (87 g, 415 mmol) was added slowly over a period of 1 h. After addition the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 24 h; and the reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water. The organic layer was separated, and the aqueous layer was extracted with DCM (2×40 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford 32 (44.5 g, yield: 99.7%) as a liquid.

Synthesis of Dodecyl 2-(dimethylamino)acetate.HCl (33, Nex-52)

A stirred solution of 32 (44.5 g, 164 mmol) in ethyl acetate/hexane (50:400 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes. The reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (5×50 mL) to afford wet dodecyl 2-(dimethylamino)acetate.HCl 33 (45 g) as a semi solid. This semi solid was taken in ethyl acetate/hexane (10:90 mL), heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained solid was filtered under nitrogen and dried under vacuum to afford dodecyl 2-(dimethylamino)acetate.HCl salt (33, Nex-52) (28 g, yield, 54.9%) as a white hygroscopic solid, mp: 65-70° C. ¹H-NMR (400 MHz, CDCl₃): δ 4.2 (t, 2H), 3.9 (s, 2H), 3.0 (s, 6H), 1.6-1.7 (m, 2H), 1.2-1.4 (m, 18H), 0.9 (t, 3H) LCMS: 272 (M⁺+1); HPLC: 99.28%.

FIG. 13A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino)acetate.HCl salt (Nex-52).

FIG. 13B is a LCMS spectrum: 272 (M⁺+1) of dodecyl 2-(dimethylamino)acetate.HCl salt.

FIG. 13C is a HPLC chromatogram of dodecyl 2-(dimethylamino)acetate.HCl salt showing a peak area of 99.28%. Methods as in FIG. 4D.

Example 15 Synthesis of Dodecyl 2-(dimethylamino)-3-methylbutanoate.HCl (Nex-53)

Synthesis of Dodecyl 2-amino-3-methylbutanoate (35)

To a stirred solution of DL-valine 34 (20 g, 170.7 mmol) in toluene (200 mL) was added 1-dodecanol 2 (28.6 g, 153 mmol) in one lot, followed by pTSA (35.7 g, 187.7 mmol). After addition, the temperature of the reaction mixture was slowly raised to reflux temperature and the water was separated azeotropically. The reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 35 (40 g, yield, 82%) as a liquid.

Synthesis of Dodecyl 2-(dimethylamino)-3-methylbutanoate (36)

To a stirred solution of 35 (40 g, 140 mmol) in DCM (500 mL) was added aqueous formaldehyde solution (37% w/v) (14.7 g, 490 mmol) in one lot at 0° C. and slowly added Na (OAc)₃BH (74.2 g, 350 mmol) over a period of 1 h. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 24 h. The reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×40 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford 36 (42 g, yield: 96%) as a liquid.

Synthesis of dodecyl 2-(dimethylamino)-3-methylbutanoate.HCl (37, Nex-53)

A stirred solution of 36 (42 g, 133 mmol) in ethyl acetate/hexane (50:150 mL) and was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (5×50 mL) to afford wet dodecyl 2-(dimethylamino) 3-methylbutanoate.HCl 37 (40 g) as a semi solid. The semi solid was taken in ethyl acetate/hexane (10:90 mL), heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained solid was filtered under nitrogen and dried under vacuum to afford dodecyl 2-(dimethylamino) 3-methylbutanoate.HCl salt (37, Nex-53) (27 g, yield: 57.6%) as a white hygroscopic solid, mp: 106-110° C. ¹H-NMR (400 MHz, CDCl₃): δ 4.3 (t, 2H), 3.6 (m, 1H), 3.0-2.8 (dd, 6H), 2.4 (q, 1H), 1.7 (m, 2H), 1.2-1.4 (m, 21H) 1.1 (d, 3H), 0.9 (t, 3H) LCMS: 314 (M⁺+1); HPLC: 99.98%.

FIG. 14A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) 3-methylbutanoate.HCl salt (Nex-53).

FIG. 14B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 14A.

FIG. 14C is a LCMS spectrum: 314 (M⁺+1) of dodecyl 2-(dimethylamino) 3-methylbutanoate.HCl salt.

FIG. 14D is a HPLC chromatogram of dodecyl 2-(dimethylamino) 3-methylbutanoate.HCl salt showing a peak area of 99.98%. Methods as in FIG. 4D.

Example 16 Synthesis of Dodecyl 2-(dimethylamino) 3-phenyl propanoate HCl (Nex-54)

Synthesis of Dodecyl 2-amino-3-phenylpropanoate (39)

To a stirred solution of DL-phenylalanine 38 (5 g, 30.26 mmol) in toluene (100 mL) was added dodecanol 2 (5.08 g, 27.24 mmol) in one lot, followed by pTSA (6.33 g, 33.29 mmol). The temperature of the reaction mixture was slowly raised to reflux temperature, and the water was separated azeotropically The reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 39 (9 g, yield: 89.1%) as a liquid.

Synthesis of dodecyl 2-(dimethylamino) 3-phenylpropanoate (40)

To a stirred solution of 39 (9 g, 26.97 mmol) in DCM (45 mL) was added aqueous formaldehyde solution (37% w/v) (2.83 g, 94.3 mmol) in one lot at 0° C. and Na (OAC)₃BH (14.29 g, 67.4 mmol) was slowly added over a period of ½ h. After the addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 24 h. The reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×40 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na2SO4, and concentrated under vacuum to afford 40 (9 g, yield: 92.7%) as a liquid.

Synthesis of dodecyl 2-(dimethylamino) 3-phenyl propanoate.HCl (41, Nex-54)

To a stirred solution of 40 (9 g, 24.8 mmol) in ethyl acetate/hexane (10:90 mL) and then cooled to 0° C. The reaction mixture was purged with dry HCl gas for 15 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (5×50 mL) to afford wet dodecyl 2-(dimethylamino) 3-phenyl propanoate. HCl 41 (9 g) as a semi solid. Above semi solid was taken in ethyl acetate/hexane (10:90 mL) and heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford dodecyl 2-(dimethylamino) 3-phenyl propanoate.HCl salt (7 g, yield: 70.07%) as a white hygroscopic solid, mp: 71-76° C. ¹H-NMR (400 MHz, CDCl₃): δ 7.4-7.2 (m, 5H), 4.1-4.0 (m, 3H), 3.8 (d, 1H), 3.1 (m, 1H), 3.0 (s, 3H), 2.8 (s, 3H) 1.4 (m, 2H), 1.35-1.0 (m, 18H), 0.9 (t, 3H) LCMS: 362 (M⁺+1); HPLC: 99.8%.

FIG. 15A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino) 3-phenyl propanoate.HCl salt (Nex-54).

FIG. 15B is a LCMS spectrum: 314 (M⁺+1) of dodecyl 2-(dimethylamino) 3-phenyl propanoate.HCl salt.

FIG. 15C is a HPLC chromatogram of dodecyl 2-(dimethylamino) 3-phenyl propanoate.HCl salt showing a peak area of 99.98%. Methods as in FIG. 4D

Example 17 Dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl (Nex-55)

Synthesis of dodecyl 2-amino-4-methylpentanoate (43)

To a stirred solution of DL-leucine 42 (20 g, 152 mmol) in toluene (400 mL) was added 1-dodecanol 2 (25.56 g, 137.2 mmol) in one lot, followed by pTSA (31.9 g, 167.7 mmol). After addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 43 (42 g, yield: 91.9%) as a liquid.

Synthesis of dodecyl 2-(dimethylamino)-4-methylpentanoate (44)

To a stirred solution of 43 (40 g, 133 mmol) in DCM (500 mL) was added aqueous formaldehyde solution (37% w/v) (14 g, 467 mmol) in one lot at 0° C. Na (OAc)₃BH (70.7 g, 333.8 mmol) was slowly added over a period of 1 h. After the addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 24 h. The reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated, and the aqueous layer was extracted with DCM (2×40 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford 44 (42 g, yield: 91%) as a liquid.

Synthesis of dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl (45, Nex-55)

A stirred solution of 44 (40 g, 121 mmol) in ethyl acetate/hexane (50:150 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (5×50 mL) to afford wet dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl 45 (40 g) as a semi solid. The semi solid was taken in ethyl acetate/hexane (10:90 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl salt (25 g, yield: 56.2%) as a white hygroscopic solid, mp: 104-109° C. ¹H-NMR (400 MHz, CDCl₃): δ 4.2 (t, 2H), 3.9 (d, 1H), 2.8-2.6 (dd, 6H), 2.0-1.8 (m, 2H), 1.7 (m, 3H), 1.4.-1.2 (m, 18H), 1.35-1.0 (m, 18H), 1.0 (dd, 6H), 0.7 (t, 3H) LCMS: 328 (M⁺+1); HPLC: 99.92%.

FIG. 16A is a ¹H-NMR spectrum of dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl salt (Nex-55).

FIG. 16B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 16A.

FIG. 16C is a LCMS spectrum: 328 (M⁺+1) of dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl salt.

FIG. 16D is a HPLC chromatogram of dodecyl 2-(dimethylamino)-4-methylpentanoate.HCl salt showing a peak area of 99.92%. Methods as in FIG. 4D.

Example 18 D-Dodecyl 2-(dimethylamino) propanoate hydrochloride (Nex-56)

Synthesis of D-Dodecyl 2-amino propanoate (47)

To a stirred solution of D-alanine 46 (15 g, 168.5 mmol) in toluene (200 mL) was added 1-dodecanol 2 (28.26 g, 151.6 mmol) in one lot, followed by pTSA (35.25 g, 185.3 mmol). After the addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 47 (42 g, yield: 97.6%) as a liquid.

Synthesis of D-Dodecyl 2-(dimethylamino) propanoate (48)

To a stirred solution of 47 (42 g, 163 mmol) in DCM (500 mL) was added aqueous formaldehyde solution (37% w/v) (17.13 g, 571 mmol) in one lot at 0° C. Na (OAC)₃BH (86.47 g, 408 mmol) was slowly added over a period of 1 h. After the addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 12 h; the reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×250 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford D-Dodecyl 2-(dimethylamino) propanoate 48 (42 g, yield: 96.9%) as a liquid.

Synthesis of D-Dodecyl 2-(dimethylamino) propanoate hydrochloride (49, Nex-56)

A stirred solution of D-Dodecyl 2-(dimethylamino) propanoate 48 (42 g, 147 mmol) in ethyl acetate/hexane/MeOH (100:100:10 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×52 mL) followed by hexane (3×25 mL) to afford D-Dodecyl 2-(dimethylamino) propanoate hydrochloride salt 49 (42 g) as a semi solid. The semi solid was taken in hexane (100 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford (R)-Dodecyl 2-(dimethylamino) propanoate hydrochloride salt 49 (27 g, yield: 57.4%) as a white hygroscopic solid, mp: 86-89° C. ¹H-NMR (400 MHz, CDCl₃): δ 4.25 (m, 2H), 4.0 (m, 1H), 3.9 (s, 6H), 1.8-1.6 (m, 5H), 1.4-1.2 (m, 18H), 0.9 (t, 3H); LCMS: 286 (M⁺+1); HPLC: 99.8%.

FIG. 17A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of (R)-dodecyl 2-(dimethylamino) propanoate hydrochloride salt (Nex-56).

FIG. 17B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 17A.

FIG. 17C is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 17A.

FIG. 17D is a LCMS spectrum: 286.26 (M⁺+1) of (R)-dodecyl 2-(dimethylamino) propanoate hydrochloride salt.

FIG. 17E is a HPLC chromatogram of (R)-dodecyl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.92%. Methods as in FIG. 4D

FIG. 17F is a HPLC chromatogram of the DL-dodecyl 2-(dimethylamino) propanoate hydrochloride salt racemate showing the separate peaks of the two stereoisomers. Methods: column: Chiralpak AD-3 (250×4.6 mm, 3 μm); mobile phase: 0.1% diethyl amine in Methanol (100:0.1); injection volume 10 μL, column temperature, 15° C., flow rate 1.0 mL/min; detection 235 nm 4 nm.

FIG. 17G is a HPLC chromatogram of D-dodecyl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.92%. Methods as in FIG. 17F, except that the detection was 235 nm 8 nm.

Example 19 L-Dodecyl 2-(dimethylamino) propanoate hydrochloride (Nex-57)

Synthesis of L-dodecyl 2-amino propanoate (51)

To a stirred solution of (S) L-alanine 50 (15 g, 168.5 mmol) in toluene (200 mL) was added 1-dodecanol 2 (28.26 g, 151.6 mmol) in one lot, followed by pTSA (35.25 g, 185.3 mmol). After the addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 51 (42 g, yield: 97.6%) as a liquid.

Synthesis of L-Dodecyl 2-(dimethylamino) propanoate (52)

To a stirred solution of 51 (42 g, 163 mmol) in DCM (500 mL) was added aqueous formaldehyde solution (37% w/v) (17.13 g, 571 mmol) in one lot at 0° C. and Na (OAC)₃BH (86.47 g, 408 mmol) was slowly added over a period of 1 h. After the addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 12 h. The reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated, and the aqueous layer was extracted with DCM (2×250 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford L-DDAIP 52 (42 g, yield: 96.9%) as a liquid.

Synthesis of L-Dodecyl 2-(dimethylamino) propanoate hydrochloride

A stirred solution of L-DDAIP 52 (42 g, 147 mmol) in ethyl acetate/hexane/MeOH (100:100:10 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×52 mL) followed by hexane (3×25 mL) to afford L-dodecyl 2-(dimethylamino) propanoate hydrochloride (53, Nex-57) salt (42 g) as a semi solid. The semi solid was taken in hexane (100 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt (27 g, yield: 57.4%) as a white hygroscopic solid, mp: 86-90° C. ¹H-NMR (400 MHz, CDCl₃): δ 4.25 (m, 2H), 4.0 (m, 1H), 3.9 (s, 6H), 1.8-1.6 (m, 5H), 1.4-1.2 (m, 18H), 0.9 (t, 3H); LCMS: 286 (M⁺+1); HPLC: 99.8%.

FIG. 18A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt (Nex-57).

FIG. 18B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 18A.

FIG. 18C is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 18A.

FIG. 18D is a LCMS spectrum: 286.28 (M⁺+1) of L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt.

FIG. 18E is a HPLC chromatogram of L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.85%. Methods as in FIG. 4D

FIG. 18F is a HPLC chromatogram of L-dodecyl 2-(dimethylamino) propanoate hydrochloride salt showing a peak area of 99.5%. Methods as in FIG. 17F.

Example 20 Dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride (Nex-58)

Synthesis of dodecyl 2-amino-2-methylpropanoate (55)

To a stirred solution of 2-aminoisobutyric acid 54 (10 g, 96.9 mmol) in toluene (200 mL) was added 1-dodecanol 2 (16.26 g, 87.27 mmol) in one lot, followed by pTSA (20.27 g, 106.6 mmol). After the addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 56 (22 g, yield: 83.65%) as a liquid.

Synthesis of dodecyl 2-(dimethylamino)-2-methylpropanoate (56)

To a stirred solution of 55 (5 g, 18.45 mmol) in DCM (200 mL) was added aqueous formaldehyde solution (37% w/v) (1.93 g, 64.5 mmol) in one lot at 0° C., and Na (OAC)₃BH (9.77 g, 46.12 mmol) was added over a period of 1 h. After the addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 24 h; the reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated, and the aqueous layer was extracted with DCM (2×40 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford dodecyl 2-(dimethylamino)-2-methylpropanoate 56 (5.0 g, yield: 90.9%) as a liquid.

Synthesis of Dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride (57, Nex-58)

A stirred solution of DDAIP Derivative (5 g, 16.7 mmol) in ethyl acetate/hexane/MeOH (25:25:5 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was flushed with ethyl acetate (3×52 mL) followed by hexane (3×25 mL) to afford Dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride (57, Nex-58) (5 g) as a semi solid. The semi solid was taken in ethyl acetate/hexane (10:10 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford Dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride (57, Nex-58) salt (3.2 g, yield: 57.1%) as a white hygroscopic solid, mp: 68-74° C. ¹H-NMR (400 MHz, CDCl₃): δ 0.82 (t, 3H), 1.3 (m, 18H), 1.7 (q, 2H), 1.8 (s, 6H), 4.2 (t, 2H); LCMS: 300 (M⁺+1); HPLC: 98.07%.

FIG. 19A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride salt (Nex-58).

FIG. 19B is a LCMS spectrum: 300 (M⁺+1) of dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride salt.

FIG. 19C is a HPLC chromatogram of dodecyl 2-(dimethylamino)-2-methylpropanoate hydrochloride salt showing a peak area of 98.07%. Methods as in FIG. 4D

Example 21 Dodecyl 2-(methylamino) propanoate hydrochloride (Nex-59)

Synthesis of dodecyl 2-bromopropanoate (59)

To a stirred solution of 1-decanol 2 (10 g, 53.7 mmol) in toluene (100 mL) was added triethylamine (7.5 mL, 53.7 mmol)) and followed by 2-bromo propionyl bromide 58 (12.7 g, 59.1 mmol) at 5-10° C. The reaction mixture was stirred for 3 hour at 55-60° C., and the reaction was monitored by TLC. The reaction mixture was quenched with saturated sodium bicarbonate solution and stirred for 15 minutes at 25-35° C. The aqueous and organic layers were separated, the combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 59 (16.19 g, yield: 94%) as a liquid.

Synthesis of dodecyl 2-(methylamino) propanoate (60)

To a stirred solution of 59 (10 g, 31.2 mmol) in acetonitrile (20 mL) was added sodium bicarbonate (2.62 g, 31.2 mmol) and followed by mono methyl amine (40% in water) (30 mL, 3 vol) at 25-30° C. The reaction mixture was stirred for 3 hour at 25-30° C.; the reaction was monitored by TLC. The solid obtained in the reaction mixture was filtered under vacuum. The solvent was concentrated, diluted with ethyl acetate/water and stirred for 15 minutes at 25-30° C. The aqueous and organic layers were separated, and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic layers were washed with brine solution, dried over Na₂SO₄ and concentrated under vacuum to afford crude 60 (7 g, yield: 82.3%) as a liquid

Synthesis of dodecyl 2-(methylamino) propanoate hydrochloride (61, Nex-59)

A stirred solution of 60 (7 g, 25.8 mmol) in hexane (30 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 10 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×25 mL) followed by hexane (5×20 mL) to afford wet dodecyl 2-(methylamino) propanoate hydrochloride salt (61, Nex-59) (7 g) as a waxy solid. The waxy solid was taken in ethyl hexane (50 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford dodecyl 2-(methylamino) propanoate hydrochloride salt (3 g, yield: 37.9%) as a white hygroscopic solid, mp: 78-83° C. ¹H-NMR (400 MHz, CDCl₃): δ 4.3-4.2 (m, 2H), 3.9 (d, 1H), 2.8 (s, 3H), 1.8-1.6 (m, 5H), 1.4-1.2 (m, 18H), 0.9 (t, 3H) LCMS: 272.31 (M⁺+1); HPLC: 98.45%.

FIG. 20A is a ¹H-NMR spectrum (400 MHz, CDCl₃) of dodecyl 2-(methylamino) propanoate hydrochloride salt (Nex-59)

FIG. 20B is a ¹H-NMR spectrum at a higher resolution to resolve the peaks of FIG. 20A.

FIG. 20C is a LCMS spectrum: 272.3 (M⁺+1) of dodecyl 2-(methylamino)-2-methylpropanoate hydrochloride salt.

FIG. 20D is a HPLC chromatogram of dodecyl 2-(methylamino) propanoate hydrochloride salt showing a peak area of 98.45%. Methods as in FIG. 4D.

Example 22 Dodecyl 2-(isopropyl amino) propanoate hydrochloride (Nex-60)

Synthesis of dodecyl 2-bromopropanoate (59)

To a stirred solution of 1-decanol 2 (10 g, 53.7 mmol) in toluene (100 mL) was added triethylamine (7.5 mL, 53.7 mmol)), followed by 2-bromo propionyl bromide 58 (12.7 g, 59.1 mmol) at 5-10° C. The reaction mixture was stirred for 3 hour at 55-60° C. and the reaction was monitored by TLC. The reaction mixture was quenched with saturated sodium bicarbonate solution and stirred for 15 minutes at 25-35° C. The aqueous and organic layers were separated, the organic layer was washed with brine, and the combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 59 (16.2 g, yield: 94%) as a liquid.

Synthesis of dodecyl 2-(isopropyl amino) propanoate (62)

To a stirred solution of 59 (10 g, 31.2 mmol) in acetonitrile (30 mL) was added sodium bicarbonate (2.62 g, 31.2 mmol) and followed by isopropyl amine (50% in water) (30 mL, 3 vol) at 25-30° C. The reaction mixture was stirred for 12 h at 55-60° C. and the reaction was monitored by TLC. The solid obtained in the reaction mixture was filtered under vacuum. The solvent was concentrated, diluted with ethyl acetate/water and stirred for 15 minutes at 25-30° C. The aqueous and organic layers were separated, and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 62 (7 g, yield: 75.2%) as a liquid.

Synthesis of dodecyl 2-(isopropyl amino) propanoate hydrochloride

A stirred solution of 62 (7 g, 23.4 mmol) in hexane (50 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 10 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was flushed with ethyl acetate (3×25 mL) followed by hexane (5×20 mL) to afford wet dodecyl 2-(isopropyl amino) propanoate hydrochloride 63 ((7 g) as a solid. The solid was taken in hexane (30 mL) and heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford Dodecyl 2-(isopropyl amino) propanoate hydrochloride salt (63, Nex-60) (5 g, yield: 64%) as a white hygroscopic solid, mp: 86-91° C. ¹H-NMR (400 MHz, CDCl₃) δ 4.3-4.2 (m, 2H), 3.9 (m, 1H), 3.5 (m, 1H), 1.9 (d, 3H), 1.7 (m, 2H), 1.6 (d, 3H), 1.45 (d, 3H), 1.4-1.2 (m, 18H), 0.9 (t, 3H). LCMS: 300.31 (M⁺+1); HPLC: 98.6%.

Example 23 Dodecyl 2-(2-hydroxyethyl) propanoate hydrochloride (Nex-61)

Synthesis of dodecyl 2-bromopropanoate (59)

To a stirred solution of 1-decanol 2 (10 g, 53.7 mmol) in toluene (100 mL) was added triethylamine (7.5 mL, 53.7 mmol)) and followed by 2-bromo propionyl bromide 58 (12.7 g, 59.1 mmol) at 5-10° C. The reaction mixture was stirred for 3 hour at 55-60° C., and the reaction was monitored by TLC. The reaction mixture was quenched with saturated sodium bicarbonate solution and stirred for 15 minutes at 25-35° C. The aqueous and organic layers were separated, the organic layer was washed with brine, and the combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 59 (16.2 g, yield: 94%) as a liquid.

Synthesis of dodecyl 2-((2-hydroxyethyl)amino) propanoate (64)

To a stirred solution of 3 (1 g, 3.2 mmol) in acetonitrile/water (5:5 mL) was added sodium bicarbonate (0.262 g 3.2 mmol) and followed by ethanol amine (0.5 mL, 0.5 vol) at 25-30° C. The reaction mixture was stirred for 12 h at RT; the reaction was monitored by TLC. The solid obtained in the reaction mixture was filtered under vacuum, the solvent concentrated, diluted with ethyl acetate/water and stirred for 15 minutes at 25-30° C. The aqueous and organic layers were separated, and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 64 (0.9 g, yield: 95%) as a liquid

Synthesis of dodecyl 2-((2-hydroxyethyl)amino) propanoate hydrochloride (65, Nex-61)

A stirred solution of 64 (0.9 g, 2.99 mmol) in hexane (10 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 10 minutes; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×25 mL) followed by hexane (5×20 mL) to afford wet dodecyl 2-((2-hydroxyethyl)amino) propanoate hydrochloride 65 (1 g) as a solid. The solid was taken in hexane (10 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford dodecyl 2-((2-hydroxyethyl)amino) propanoate hydrochloride salt 65 (0.52 g, yield: 52%) as a white hygroscopic solid, mp: 134-139° C. ¹H-NMR (400 MHz, CDCl₃): δ 4.3-4.2 (m, 2H), 4.1-4.0 (m, 3H), 3.3 (m, 2H), 1.8-1.6 (m, 5H), 1.4-1.2 (m, 18H), 0.9 (t, 3H), LCMS: 302.47 (M⁺+1); HPLC: 93.9%.

Example 24 Dodecyl 2-((2-(diethylamino)ethyl)amino) propanoate hydrochloride (Nex-62)

Synthesis of Dodecyl 2-bromopropanoate (59)

To a stirred solution of 1-decanol 2 (10 g, 53.7 mmol) in toluene (100 mL) was added triethylamine (7.5 mL, 53.7 mmol)) followed by 2-bromo propionyl bromide 58 (12.7 g, 59.1 mmol) at 5-10° C. The reaction mixture was stirred for 3 hours at 55-60° C.; the reaction was monitored by TLC. The reaction mixture was quenched with saturated sodium bicarbonate solution and stirred for 15 minutes at 25-35° C. The aqueous and organic layers were separated, the organic layer was washed with brine, and the combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 59 (16.2 g, yield: 94%) as a liquid.

Synthesis of dodecyl 2-((2-(diethyl amino) ethyl)amino) propanoate (66)

To a stirred solution of 59 (1 g, 3.2 mmol) in acetonitrile/water (5:5 mL) was added sodium bicarbonate (0.262 g 3.2 mmol) and followed by N,N-diethyl-1,2-diamine (0.5 mL, 0.5 vol) at 25-30° C. The reaction mixture was stirred for 12 h at RT; the reaction was monitored by TLC. The solid obtained in the reaction mixture was filtered under vacuum. The solvent was concentrated, diluted with ethyl acetate/water and stirred for 15 minutes at 25-30° C. The aqueous and organic layers were separated, and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 66 (0.9 g, yield: 95%) as a liquid

Synthesis of dodecyl 2-((2-(diethyl amino) ethyl)amino) propanoate hydrochloride

A stirred solution of 66 (0.9 g, 2.99 mmol) in hexane (10 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 10 minutes; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×25 mL) followed by hexane (5×20 mL) to afford wet dodecyl 2-((2-(diethyl amino) ethyl)amino) propanoate hydrochloride (67, Nex-62) (1 g) as a solid. The solid was taken in hexane (10 mL), heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford dodecyl 2-((2-(diethyl amino) ethyl)amino) propanoate hydrochloride salt 67 (0.25 g, yield: 52%) as a white hygroscopic solid. ¹H-NMR (400 MHz, CDCl₃) δ 4.3-4.2 (m, 2H), 4.1-3.9 (m, 3H), 3.8-3.6 (d, 2H), 3.4-3.2 (m, 4H), 1.8-1.6 (m, 5H), 1.5 (d, 6H), 1.4-1.2 (m, 18H), 0.9 (t, 3H). LCMS: 357.59 (M⁺+1); q-¹HNMR: 96.94%.

Example 25 Synthesis of Tridecan-2-yl 2-(dimethylamino) propanoate hydrochloride (Nex-64)

Synthesis of Tridecan-2-yl 2-aminopropanoate (69)

To a stirred solution of DL-alanine 1 (15 g, 168.5 mmol) in toluene (300 mL) was added 2-tridecanol 68 (30.3 g, 151.68 mmol) in one lot, followed by pTSA (35.26 g, 185.38 mmol). After the addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 69 (36 g, yield: 78.9%) as a liquid.

Synthesis of tridecan-2-yl 2-(dimethylamino) propanoate (70)

To a stirred solution of 69 (5 g, 18.4 mmol) in DCM (200 mL) was added aqueous formaldehyde solution (37% w/v) (1.93 g, 64.4 mmol) in one lot at 0° C. and Na (OAC)₃BH (9.76 g, 46.06 mmol) was slowly added over a period of 1 h. After the addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 24 h. The reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated, and the aqueous layer was extracted with DCM (2×30 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford tridecan-2-yl 2-(dimethylamino) propanoate (70) (5 g, yield: 90.9%) as a liquid.

Synthesis of tridecan-2-yl 2-(dimethylamino) propanoate hydrochloride (71, Nex-64)

A stirred solution of 70 (5 g, 16.7 mmol) in ethyl acetate/hexane/MeOH (25:25:5 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was flushed with ethyl acetate (3×25 mL) followed by hexane (3×25 mL) to afford tridecan-2-yl 2-(dimethylamino) propanoate hydrochloride salt 71 (5 g) as a liquid. The liquid taken in ethyl acetate/hexane (10:10 mL), heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained semi solid was filtered under nitrogen. The obtained wet solid was taken in hexane (25 mL) and heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT over a period of 12 h and then to 0° C. The obtained solid was filtered under nitrogen, and dried under vacuum to afford tridecan-2-yl 2-(dimethylamino) propanoate hydrochloride (71, Nex-64) (2.5 g, yield: 44.6%) as a white hygroscopic solid, mp: 88-94° C. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.8 (t, 3H), 1.3 (m, 18H), 1.3 (d, 3H), 1.45 (m, 1H), 1.6 (m, 1H), 1.7 (d, 3H), 2.9 (s, 6H); LCMS: 300 (M⁺+1); HPLC: 99.62%.

Example 26 Synthesis of 2-Methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride (Nex-65)

Synthesis of 2-methyltridecan-2-ol (72)

To a cooled solution of methyl laurate 72 (31 g, 144 mmol) in THF (600 mL) in an ice-water bath was added a solution of 3M methyl magnesium bromide in ether (100 mL, 303 mmol) drop wise via cannula and the stirred reaction mixture was allowed to warm ambient temperature and stirred for 12 hours, and the reaction mixture was monitored by TLC. The reaction mixture was poured into 500 mL of 2M sulfuric acid solution and was extracted with ethyl acetate. The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to obtained crude. The obtained crude was purified by column chromatography (5%-10% Ethyl acetate in hexane) to afford 73 (20 g yield: 64.9%) as a liquid.

Synthesis of 2-methyltridecan-2-yl 2-bromopropanoate (74)

To a stirred solution of 2-methyltridecan-2-ol 73 (5 g, 23.4 mmol) in Toluene (50 mL) was added triethylamine (3.29 mL, 23.4 mmol)) and followed by 2-bromopropionyl bromide 58 (5.11 g, 23.7 mmol) at 5-10° C. The reaction mixture was stirred for 3 hours at 55-60° C.; the reaction was monitored by TLC. The reaction mixture was quenched with saturated sodium bicarbonate solution and stirred for 15 minutes at 25-35° C. The aqueous and organic layers were separated, the organic layer was washed with brine, and the combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 74 (5 g, yield: 61.27%) as a liquid.

Synthesis of 2-methyltridecan-2-yl 2-(dimethylamino) propanoate (75)

To a stirred solution of 74 (2 g, 5.7 mmol) in acetonitrile (10 mL) was added sodium bicarbonate (0.48 g, 5.74 mmol) and followed by dimethyl amine (40% in water) (10 mL, 88.8 mmol) at 25-30° C. The reaction mixture was stirred for 3 hours at 25-30° C.; the reaction was monitored by TLC. The solid obtained in the reaction mixture was filtered under vacuum. The solvent was concentrated, diluted with ethyl acetate/water and stirred for 15 minutes at 25-30° C. The aqueous and organic layers were separated, and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 75 (1.49 g, yield: 83%) as a liquid.

Synthesis of 2-methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride (76, Nex-65)

A stirred solution of 75 (1.49 g, 4.74 mmol) in ethyl acetate/hexane (1:9 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 10 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was flushed with ethyl acetate (3×25 mL) followed by hexane (5×20 mL) to afford wet 2-methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride (76, Nex-65) (1.4 g) as a semi solid. Above semi solid was taken in ethyl acetate/hexane (1:9 mL) and heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford 2-methyltridecan-2-yl 2-(dimethylamino) propanoate hydrochloride salt (0.49 g, yield: 29.5%) as a white hygroscopic solid, mp: 100-106° C. ¹H-NMR (400 MHz, CDCl₃): δ 3.9 (m, 1H), 2.9 (s, 6H), 1.8-1.6 (m, 5H), 1.5 (s, 6H), 1.3-1.2 (m, 18H), 0.9 (t, 3H). LCMS: 314 (M⁺+1); HPLC: 95.7%.

Example 27 Synthesis of Tetradecyl-2-N,N-dimethylaminopropionate hydrochloride (Nex-66)

Synthesis of tetradecyl 2-aminopropanoate (78)

To a stirred solution of DL-alanine 1 (10 g, 112.35 mmol) in toluene (200 mL) was added 1-Tetradecanol (21.6 g, 101.12 mmol) in one lot, followed by pTSA (23.5 g, 123.58 mmol). After addition the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 78 (30 g, yield: 93.75%) as a liquid.

Synthesis of tetradecyl 2-(dimethylamino) propanoate (79)

To a stirred solution of 78 (30 g, 105.26 mmol) in DCM (200 mL) was added aqueous formaldehyde solution (37% w/v) (11.05 g, 368.4 mmol) in one lot at 0° C. and slowly added Na(OAc)₃BH (55.77 g, 263.15 mmol) over a period of 1 h. After the addition, the temperature of the reaction mixture was slowly raised to RT, stirred at RT for 24 h, and the reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×40 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford 79 (30 g, yield: 90.9%) as a liquid.

Synthesis of Tetradecyl-2-N,N-dimethylaminopropionate hydrochloride (80, Nex-66)

A stirred solution of 79 (30 g, 95.84 mmol) in ethyl acetate/hexane/MeOH (100:100:10 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (5×50 mL) to afford tetradecyl-2-N,N-dimethylaminopropionate HCl salt 80 (35 g) as a semi solid. Above semi solid was taken in ethyl acetate/hexane (100:100 mL) and heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The reaction mixture was filtered under nitrogen and dried under vacuum to afford Tetradecyl-2-N,N-dimethylaminopropionate. HCl salt (23 g, yield: 68.6%) as a white hygroscopic solid, mp: 93-96.5° C. ¹H-NMR (400 MHz, CDCl₃): δ 4.3 (m, 2H), 4.0 (q, 1H), 2.9 (s, 6H), 1.8-1.6 (m, 5H), 1.4.-1.2 (m, 22H), 0.9 (t, 3H); LCMS: 314 (M⁺+1); HPLC: 99.7%.

Example 28 Synthesis of Undecyl-2-N,N-dimethylaminopropionate hydrochloride (Nex-67)

Synthesis of undecyl 2-aminopropanoate (82)

To a stirred solution of DL-alanine 1 (15 g, 168.5 mmol) in toluene (300 mL) was added 1-undecanol 81 (26.22 g, 151.68 mmol) in one lot, followed by pTSA (35.38 g, 185.35 mmol). After the addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 82 (40 g, yield: 97.56%) as a liquid.

Synthesis of undecyl 2-(dimethylamino) propanoate (83)

To a stirred solution of 82 (40 g, 164.39 mmol) in DCM (200 mL) was added aqueous formaldehyde solution (37% w/v) (17.26 g, 575.39 mmol) in one lot at 0° C. and slowly added Na (OAC)₃BH (9.76 g, 410.99 mmol) over a period of 1 h. After the addition, the temperature of the reaction mixture was slowly raised tort, stirred at RT for 24 h; the reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×30 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford 83 (42 g, yield: 95.45%) as a liquid.

Synthesis of undecyl-2-N,N-dimethylaminopropionate hydrochloride (84, Nex-67)

To a stirred solution of 83 (42 g, 154.8 mmol) in ethyl acetate/hexane/MeOH (25:25:5 mL) and then cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes; the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was flushed with ethyl acetate (3×25 mL) followed by hexane (3×25 mL) to afford undecyl-2-N,N-dimethylaminopropionate.HCl salt 84 (40 g) as a liquid. The liquid taken in ethyl acetate/hexane (100:100 mL) and heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained semi solid was filtered under nitrogen. The obtained wet solid was taken in hexane (50 mL) and heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT over a period of 12 h and then to 0° C. The obtained waxy solid was filtered under nitrogen, dried under vacuum to afford undecyl-2-N,N-dimethylaminopropionate.HCl salt 84 (27 g, yield: 59.94%) as a waxy hygroscopic solid. ¹H-NMR (400 MHz, DMSO-d₆): δ 4.3-4.15 (m, 3H), 2.9 (s, 6H), 1.7 (t, 3H), 1.55 (d, 3H), 1.4-1.2 (m, 16H), 1.9 (m, 3H); LCMS: 272 (M⁺+1); HPLC: 99.6%.

Example 29 Synthesis of Decyl-2-N,N-dimethylaminopropionate hydrochloride (Nex-68)

Synthesis of decyl 2-aminopropanoate (86)

To a stirred solution of DL-alanine 1 (15 g, 168.5 mmol) in toluene (300 mL) was added 1-decanol 85 (23.9 g, 151.68 mmol) in one lot, followed by pTSA (35.26 g, 185.38 mmol). After the addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 86 (35 g, yield: 90.6%) as a liquid.

Synthesis of decyl 2-(dimethylamino) propanoate (87)

To a stirred solution of 86 (35 g, 152.8 mmol) in DCM (200 mL) was added aqueous formaldehyde solution (37% w/v) (16.04 g, 534.9 mmol) in one lot at 0° C. Na (OAc)₃BH (80.98 g, 382.09 mmol) was slowly added over a period of 1 h. After addition, the temperature of the reaction mixture was slowly raised to RT and stirred at RT for 24 h. The reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×30 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford 87 (38.4 g, yield: 97.95%) as a liquid.

Synthesis of decyl-2-N,N-dimethylaminopropionate hydrochloride (88, Nex-68)

A stirred solution of 87 (38.4 g, 149.4 mmol) in ethyl acetate/hexane/MeOH (100:100:10 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (3×50 mL) to afford decyl-2-N,N-dimethylaminopropionate.HCl salt 88 (35 g) as a wet solid. The wet solid was taken in ethyl acetate/hexane (100:100 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained semi solid was filtered under nitrogen. The obtained wet solid was taken in hexane (50 mL) and heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT over a period of 12 h and then to 0° C. The obtained solid was filtered under nitrogen, dried under vacuum to afford Decyl-2-N,N-dimethylaminopropionate. HCl salt 88 (26 g, yield: 59.3%) as a white hygroscopic solid, mp: 77-82° C. ¹H-NMR (400 MHz, DMSO-d₆): δ 4.3 (m, 2H), 4.1 (q, 1H), 3 (s, 6H), 1.8-1.6 (m, 5H), 1.4-1.2 (m, 14H), 0.9 (t, 3H); LCMS: 258 (M⁺+1); HPLC: 99.18%

Example 30 Synthesis of Tridecyl-2-N,N-dimethylaminopropionate (Nex-69)

Synthesis of Tridecyl 2-aminopropanoate (90)

To a stirred solution of DL-alanine 1 (15 g, 168.5 mmol) in toluene (300 mL) was added 1-tridecanol 89 (30.39 g, 151.68 mmol) in one lot, followed by pTSA (35.26 g, 185.38 mmol). After the addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, the obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 90 (38 g, yield: 83.15%) as a liquid.

Synthesis of tridecyl 2-(dimethylamino) propanoate (91)

To a stirred solution of 90 (38 g, 140 mmol) in DCM (200 mL) was added aqueous formaldehyde solution (37% w/v) (14.7 g, 490.1 mmol) in one lot at 0° C. Na (OAc)₃BH (74 g, 350 mmol) was slowly added over a period of 1 h. After addition, the temperature of the reaction mixture was slowly raised to room temperature and stirred at RT for 24 h. The reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated, and the aqueous layer was extracted with DCM (2×30 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford 91 (40 g, yield: 95.4%) as a liquid.

Synthesis of tridecyl-2-N,N-dimethylaminopropionate hydrochloride (92, Nex-69)

A stirred solution of 91 (40 g, 133.6 mmol) in ethyl acetate/hexane/MeOH (100:100:10 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (3×50 mL) to afford tridecyl-2-N,N-dimethylaminopropionate.HCl salt 92 (38 g) as a liquid. The liquid was taken in ethyl acetate/hexane (100:100 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained semi solid was filtered under nitrogen. The obtained wet solid was taken in hexane (50 mL), heated to reflux, stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT over a period of 12 h and then to 0° C. The obtained solid was filtered under nitrogen, dried under vacuum to afford tridecyl-2-N,N-dimethylaminopropionate.HCl salt 92 (22 g, yield: 49%) as a white hygroscopic solid. ¹H-NMR (400 MHz, DMSO-d₆): δ 4.3-4.2 (m, 2H), 4.1 (q, 1H), 3 (s, 6H), 1.8-1.6 (m, 5H), 1.4-1.2 (m, 20H), 0.9 (t, 3H); LCMS: 300 (M⁺+1); HPLC: 99.89%.

Example 31 Synthesis of Octyl-2-N,N-dimethylaminopropionate hydrochloride (Nex-70)

Synthesis of octyl 2-aminopropanoate (94)

To a stirred solution of DL-alanine 1 (20 g, 224.7 mmol) in toluene (300 mL) was added 1-octanol 94 (26.08 g, 202.24 mmol) in one lot, followed by pTSA (47.02 g, 247.19 mmol). After the addition, the temperature of the reaction mixture was slowly raised to reflux temperature, the water was separated azeotropically, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum. The obtained residue was taken in ethyl acetate (200 mL) and washed with aqueous 5% Na₂CO₃ (3×50 mL) followed by brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford 94 (40 g, yield: 89.08) as a liquid.

Synthesis of octyl 2-(dimethylamino) propanoate (95)

To a stirred solution of 94 (40 g, 200 mmol) in DCM (200 mL) was added aqueous formaldehyde solution (37% w/v) (21 g, 700 mmol) in one lot at 0° C. Na (OAc)₃BH (106 g, 500 mmol) was slowly added over a period of 1 h. After addition, the temperature of the reaction mixture was slowly raised to room temperature and stirred at RT for 24 h. The reaction mixture was monitored by TLC. The reaction mixture was quenched with ice-cold water, the organic layer was separated and the aqueous layer was extracted with DCM (2×30 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄, and concentrated under vacuum to afford 95 (42 g, yield: 92.1%) as a liquid.

Synthesis of octyl-2-N,N-dimethylaminopropionate hydrochloride (96, Nex-70)

A stirred solution of 95 (42 g, 184.2 mmol) in ethyl acetate/hexane/MeOH (100:100:10 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 30 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×50 mL) followed by hexane (3×50 mL) to afford octyl-2-N,N-dimethylaminopropionate. HCl salt 96 (40 g) as a liquid. The liquid was taken in ethyl acetate/hexane (100:100 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained semi solid was filtered under nitrogen. The obtained wet solid was taken in hexane (100 mL, heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT over a period of 12 h and then to 0° C. The obtained solid was filtered under nitrogen, dried under vacuum to afford octyl-2-N,N-dimethylaminopropionate.HCl salt (96, Nex-70) (30 g, yield: 62.5%) as a hygroscopic waxy solid. ¹H-NMR (400 MHz, DMSO-d₆): δ 4.3-4.1 (m, 3H), 3 (s, 6H), 1.8-1.6 (m, 5H), 1.4-1.2 (m, 10H), 0.9 (t, 3H); LCMS: 230 (M⁺+1); HPLC: 99.56%.

Example 32 Synthesis of Tridecan-2-yl 2-(dimethylamino) 2-methyl propanoate hydrochloride (Nex-71)

Synthesis of tridecan-2-yl 2-bromopropanoate (98)

To a stirred solution of tridecan-2-ol 68 (5 g, 24.9 mmol) in toluene (50 mL) was added triethylamine (3.5 mL, 27.45 mmol)) and followed by 2-bromo-2-methylpropionyl bromide 97 (6.31 g, 27.45 mmol) at 5-10° C. The reaction mixture was stirred for 3 hour at 55-60° C., and the reaction was monitored by TLC. The reaction mixture was quenched with saturated sodium bicarbonate solution and stirred for 15 minutes at 25-35° C. The aqueous and organic layers were separated. The organic layer was washed with brine, and the combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford crude 98 (8.1 g, yield: 96%) as a liquid.

Synthesis of tridecan-2-yl 2-(dimethylamino) 2-methyl propanoate (99)

To a stirred solution of 98 (8.1 g, 23.1 mmol) in acetonitrile (50 mL) was added sodium bicarbonate (1.94 g, 23.1 mmol) and followed by dimethyl amine (40% in water) (50 mL, 444 mmol) at 25-30° C. The reaction mixture was stirred for 3 hour at 25-30° C. and the reaction was monitored by TLC. The reaction mixture was filtered under vacuum, the solvent was concentrated, diluted with ethyl acetate/water and stirred for 15 minutes at 25-30° C. The aqueous and organic layers were separated and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic layers were washed with brine solution. The organic layer was dried over Na₂SO₄ and concentrated under vacuum to afford a crude product, which was purified by column chromatography to afford 99 (7 g, yield: 95.8%) as a liquid. ¹H-NMR (400 MHz, CDCl₃) δ 4.9 (m, 1H), 2.3 (s, 3H), 2.2 (s, 3H), 1.7-1.5 (m, 2H), 1.4-1.1 (m, 27H), 0.9 (t, 3H). LCMS: 314 (M⁺+1); HPLC: 92.4%.

Synthesis of tridecan-2-yl 2-(dimethylamino) 2-methyl propanoate hydrochloride (100, Nex-71)

A stirred solution of 99 (7 g, 22.3 mmol) in ethyl acetate/hexane (1:9 mL) was cooled to 0° C. The reaction mixture was purged with dry HCl gas for 10 minutes, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was flushed with ethyl acetate (3×25 mL) followed by hexane (5×20 mL) to afford wet tridecan-2-yl 2-(dimethylamino) 2-methyl propanoate hydrochloride 100 (7 g) as a waxy solid. The waxy solid was taken in ethyl acetate/hexane (1:9 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to RT and then to 0° C. The obtained waxy solid was not filterable, and was dried under vacuum to afford tridecan-2-yl 2-(dimethylamino) 2-methyl propanoate hydrochloride salt 100 (5.6 g, yield: 72%) as a white waxy hygroscopic semi solid. ¹H-NMR (400 MHz, CDCl₃): δ 5.0 (m, 1H), 2.9-2.7 (m, 6H), 1.8 (m, 6H), 1.6-1.4 (m, 2H), 1.4-1.2 (m, 21H), 0.9 (t, 3H). LCMS: 314 (M⁺+1); HPLC: 94.36%.

Example 33 Minimum Inhibitory Concentration Assays and Time Kill Studies

Thirty compounds were tested for antimicrobial activity using standard procedures and standard strains for both the minimum inhibitory concentration (MIC) assay and a time kill study. Both the MIC assay and the time kill study were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Three bacteria strains, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213), and two fungus strains, Candida albicans (ATCC 90029) and Aspergillus niger (CMCC 98003), were used in the experiments. The strains used in the standard tests of Examples 33, 34 and 35 are listed in Table 2, below.

TABLE 2 Standard Strains of Pathogens Used For Testing Pathogen (Strain) Comments from ATCC Catalog Description Acinetobacter baumannii (ATCC 19606) Quality control strain Acinetobacter iwoffi (ATCC 15309) Type strain Aspergillus niger (CMCC 98003) Bacillus subtilis (ATCC 6633) Quality control strain, testing antibacterial activity Burkholderia cepacia (ATCC 25416) Quality control strain, assay of antimicrobial preservatives Candida albicans (ATCC 10231) Quality control strain, testing fungicides Candida albicans (ATCC 90029) Susceptibility testing of antifungal agents Corynebacterium jeikeium (ATCC 43734) Type strain Enterobacter aerogenes (ATCC 13048) Quality control strain, assay of antimicrobial preservatives Enterobacter cloacae (ATCC 13047) Quality control strain Enterococcus faecalis (ATCC 51299) Quality control strain, low-level vancomycin-resistant, VanB Enterococcus faecalis (ATCC 19433) Quality control strain Enterococcus faecium (ATCC 19434) Testing antimicrobial hand washing formulations Escherichia coli (ATCC 11229) Testing antimicrobial hand washing formulations Escherichia coli (ATCC 25922) Quality control strain Haemophilus influenza (ATCC 19418) Biovar III reference strain, media testing Klebsiella oxytoca (ATCC 43165) Quality control strain Klebsiella pneumonia (ATCC 11296) Type strain Micrococcus luteus (ATCC 4698) Type strain, quality control strain Proteus mirabilis (ATCC 7002) Quality control strain Pseudomonas aeruginosa (ATCC 9027) Quality control strain, assay of antimicrobial preservatives Pseudomonas aeruginosa (ATCC 27853) Quality control strain, susceptibility testing Serratia marcescens (ATCC 14756) Sterility assurance, testing antimicrobial agent, testing antimicrobial hand washing formulations Staphylococcus aureus (ATCC 6538) Quality control strain, testing antimicrobial agents, hand washing formulations, disinfectants, testing sanitizers, bactericides Staphylococcus aureus (MRSA) (ATCC 33592) Gentamicin- and methicillin-resistant Staphylococcus aureus (ATCC 29213) Quality control strain, susceptibility testing Staphylococcus epidermidis (ATCC 12228) Quality control strain, inhibition testing, susceptibility testing Staphylococcus haemolyticus (ATCC 29970) Type strain, quality control strain Staphylococcus hominis (ATCC 27844) Type strain, Streptococcus pneumonia (ATCC 6303) Media testing Staphylococcus saprophyticus (ATCC 15305) Type strain, quality control Streptococcus pyogenes (ATCC 19615) Quality control strain, control strain for Streptococcus Group A

Reagents included MH agar (HKM, 028050), MHB base (OXOID, CM0405), RPMI 1640 (Invitrogen, 31800-022), PDA (KAYON, P0185), YM agar (KAYON, P0271), MOPS (3-(N-morpholino)propanesulfonic acid, Sigma M3183), Amikacin USP (1019508), Ceftazidime Pentahydrate USP (1098130), Amphotericin B (amresco 0414), and Fluconazole (TCI YY10840).

MIC values were tested using six two-fold compound dilutions in duplicate with the dilution range of 12.5 mg/mL to 0.39 mg/mL. For compounds with MIC value lower than 0.39 mg/mL, MIC test was repeated using further diluted compound ranges.

Time kill studies for all compounds with an MIC value lower than 12.5 mg/mL were performed at 1×MIC concentration for four time points (0, 30 sec, 1 minute, and 5 minutes). The viable counts in Log₁₀ CFU/mL at each time point were recorded.

Preparation of cultures. Before the experiment, two days for the bacteria strains and five days for fungus strains, an aliquot was removed from the cultures frozen at −80° C. Medium was added to the surface of an appropriate agar plate and streaked with the aliquot. The plate was incubated for 20 to 24 hrs at 35±2° C. for bacteria or for 5 days at 26-30° C. in incubator for fungus. From the resulting growth on each plate, one isolated colony of similar morphology was selected and re-streaked onto a fresh agar plate using a sterile disposable loop. The plate was incubated 20 to 24 hrs at 35±2° C. or for 5 days at 26-30° C. in an incubator.

Preparation of Assay Plates with Medium and Drugs. On the day of the assay, CAMHB medium or RPMI 1640 medium was removed from 4° C. storage and allowed to reach room temperature. Ninety microliters (μL) of room temperature CAMHB or RPMI 1640 (supplemented with the appropriate concentration of DMSO, if required) were added to rows B-G of each 96-well microtiter plate. Using a pipette, duplicate 180 μL aliquots of each drug stock solution were added to column 1 (e.g. A1 and B1 or C1 and D1 or E1 and F1 or G1 and H1). Using a multichannel pipette or the Provision 2000 liquid handler, serial 2-fold dilutions (90 μL) were performed in each row across the plate to row G. Eighty μL of compounds dilutions were transferred to a 96-deep well plate. Medium, 1,520 μL of CAMHB or RPMI 1640, was added to each well. The contents of the wells were mixed thoroughly. A 50 μL aliquot of each of the resulting mixtures was transferred to a 96-well assay plate.

For bacteria, a sterile inoculating loop was used to transfer growth from an agar plate culture into about 5 mL of test medium. Using a turbidity meter, this inoculum was adjusted so that its density (600 nm) was equivalent to a 0.5 McFarland barium sulfate standard (0.08-0.13). The resulting suspension was diluted 1:100 into medium to obtain two times test inoculums.

For Candida albicans, a sterile inoculating loop was used to transfer growth from an agar plate culture into a tube with RPMI1640 medium. This inoculum was adjusted so that its density at 530 nm was in the ranges from 0.08 to 0.13. The resulting suspension was diluted 1:50 and further diluted 1:20 with the medium to obtain two times test inoculums.

For Aspergillus niger, sporulating colonies were covered with RPMI 1640, and a suspension was prepared by gently probing the colonies with the tip of a transfer pipette. The suspension was transferred to a sterile tube and mixed with a vortex mixer for 15 seconds. The densities at 530 nm were adjusted to 0.09-0.13 for each sample. The resulting suspension was diluted 1:50 with the medium to obtain two times test inoculums. Fifty μL/well of this inoculum suspension was added to wells of drug-containing plates (1:2 dilution). Plates were incubated at 35° C. for the appropriate times. The MIC was read and recorded according to CLSI guidelines.

Time kill studies. Strain suspensions were prepared as described above for the MIC test, and strain suspensions and compound solutions were mixed in the same volume. Samples were removed at specified times including 0 min, 30 sec, 1 min and 5 mins, were diluted in the test medium and streaked on agar. Colonies were counted after 24 to 48 hrs of incubation.

The MIC assay was performed using six 2-fold decremental compound dilutions in duplicate with the dilution range of 12.5 mg/mL to 0.39 mg/mL. For compounds with MIC value lower than 0.39 mg/mL, the MIC test was repeated using further diluted compound ranges. The MIC values for thirty compounds against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus niger were recorded and are presented in Table 3 and Table 4. The time kill study was performed at 1×MIC concentration for compounds with MIC values lower than 12.5 mg/mL. The viable counts in Log₁₀ CFU/mL for above five microorganisms were recorded, and are presented in Table 5 to Table 9 below.

TABLE 3 MIC (mg/mL) values for two reference compounds and thirty test compounds against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa n = 1 n = 2 compound E. coli S. aureus P. aeruginosa E. coli S. aureus P. aeruginosa Amikacin 0.002 0.002 0.002 0.002 0.002 0.002 Ceftazidime 0.0005 0.008 0.002 0.0005 0.016 0.002 Nex-01 3.13 3.13 >12.5 3.13 3.13 >12.5 Nex-03 1.56 1.56 3.13 1.56 1.56 3.13 Nex-05 3.13 3.13 6.25 3.13 1.56 6.25 Nex-07 0.78 1.56 3.13 1.56 0.78 3.13 Nex-15 >12.5 0.39 >12.5 >12.5 0.2 >12.5 Nex-16 3.13 3.13 12.5 3.13 3.13 12.5 Nex-20 1.56 3.13 6.25 1.56 1.56 6.25 Nex-22 3.13 3.13 6.25 3.13 3.13 6.25 Nex-30 3.13 3.13 6.25 3.13 3.13 6.25 Nex-32 3.13 1.56 6.25 3.13 1.56 6.25 Nex-46 3.13 3.13 6.25 3.13 3.13 6.25 Nex-52 1.56 0.2 6.25 1.56 0.1 6.25 Nex-53 6.25 6.25 6.25 6.25 6.25 6.25 Nex-54 >12.5 >12.5 >12.5 12.5 12.5 12.5 Nex-55 6.25 6.25 6.25 6.25 6.25 6.25 Nex-56 3.13 3.13 6.25 3.13 3.13 6.25 Nex-57 3.13 3.13 6.25 3.13 3.13 6.25 Nex-58 3.13 3.13 6.25 3.13 1.56 6.25 Nex-59 0.05 0.05 3.13 0.05 0.05 3.13 Nex-60 3.13 1.56 6.25 3.13 1.56 6.25 Nex-61 0.78 0.05 6.25 1.56 0.05 6.25 Nex-62 0.1 0.025 6.25 0.1 0.025 6.25 Nex-64 3.13 3.13 6.25 3.13 3.13 6.25 Nex-65 3.13 3.13 6.25 3.13 3.13 6.25 Nex-66 3.13 3.13 6.25 3.13 3.13 6.25 Nex-67 1.56 3.13 6.25 1.56 1.56 6.25 Nex-68 0.78 3.13 3.13 1.56 3.13 3.13 Nex-69 3.13 3.13 6.25 3.13 3.13 6.25 Nex-70 0.78 3.13 3.13 0.78 3.13 3.13 Nex-88 3.13 3.13 6.25 3.13 3.13 6.25

TABLE 4 MIC (mg/mL) values for two reference compounds and thirty test compounds against Candida albicans and Aspergillus niger Candida albicans Aspergillus niger Compound n = 1 n = 2 n = 1 n = 2 Amphotericin B 0.0005 0.0005 2 2 Fluconazole 0.00025 0.00025 >4 >4 Nex-01 >12.5 >12.5 >12.5 >12.5 Nex-03 12.5 6.25 >12.5 >12.5 Nex-05 >12.5 >12.5 >12.5 >12.5 Nex-07 12.5 6.25 12.5 12.5 Nex-15 0.05 0.05 12.5 >12.5 Nex-16 >12.5 12.5 >12.5 >12.5 Nex-20 12.5 12.5 >12.5 >12.5 Nex-22 >12.5 >12.5 >12.5 >12.5 Nex-30 >12.5 >12.5 >12.5 >12.5 Nex-32 >12.5 >12.5 >12.5 >12.5 Nex-46 >12.5 >12.5 >12.5 >12.5 Nex-52 0.05 0.05 3.13 3.13 Nex-53 >12.5 >12.5 >12.5 >12.5 Nex-54 6.25 6.25 12.5 12.5 Nex-55 >12.5 >12.5 >12.5 >12.5 Nex-56 >12.5 >12.5 >12.5 >12.5 Nex-57 >12.5 >12.5 >12.5 >12.5 Nex-58 >12.5 >12.5 >12.5 >12.5 Nex-59 0.025 0.025 0.05 0.05 Nex-60 12.5 12.5 >12.5 >12.5 Nex-61 0.0125 0.0125 0.05 0.025 Nex-62 0.00625 0.00625 0.0125 0.0125 Nex-64 >12.5 >12.5 >12.5 >12.5 Nex-65 >12.5 >12.5 >12.5 >12.5 Nex-66 >12.5 >12.5 >12.5 >12.5 Nex-67 6.25 6.25 12.5 12.5 Nex-68 0.39 0.2 3.13 3.13 Nex-69 12.5 12.5 >12.5 >12.5 Nex-70 0.78 0.78 12.5 12.5 Nex-88 >12.5 >12.5 >12.5 >12.5

TABLE 5 Viable counts in Log₁₀ CFU/mL for Escherichia coli time kill study compound 0 0.5 min 1 min 5 min Nex-01 5.62 5.73 5.39 <3.52 Nex-03 4.92 4.90 4.18 3.82 Nex-05 5.77 <4.0 <3.70 <3.52 Nex-07 5.88 6.02 6.02 5.68 Nex-16 5.94 5.97 5.81 5.16 Nex-20 5.86 5.84 5.92 4.37 Nex-22 5.78 4.6 <3.70 <3.52 Nex-30 5.83 5.46 3.7 <3.52 Nex-32 6.05 5.77 4.7 <3.52 Nex-46 5.87 5.74 5.91 3.97 Nex-52 5.95 6.12 5.86 5.24 Nex-53 5.64 5.72 5.71 <3.52 Nex-55 5.68 5.71 5.51 4.6 Nex-56 5.92 <4.0 <3.70 <3.52 Nex-57 5.78 <4.0 <3.70 <3.52 Nex-58 5.91 <4.0 <3.70 <3.52 Nex-59 5.34 5.04 5.11 4.56 Nex-60 5.88 <4.0 3.7 <3.52 Nex-61 5.78 5.54 5.61 4.9 Nex-62 4.82 4.78 5.02 3.82 Nex-64 5.71 5.73 5.7 <3.52 Nex-65 5.68 5.72 5.97 4.22 Nex-66 5.7 5.91 6.03 5.81 Nex-67 5.64 5.81 5.28 3.82 Nex-68 5.88 5.53 4.3 <3.52 Nex-69 6.25 5.72 5.54 <3.52 Nex-70 5.0 <4.0 <3.70 <3.52 Nex-88 5.71 <4.0 <3.70 <3.52

TABLE 6 Viable counts in Log₁₀ CFU/mL for Staphylococcus aureus time kill study compound 0 0.5 min 1 min 5 min Nex-01 6.1 6.22 6.17 6.14 Nex-03 6.11 6.1 6.12 6.11 Nex-05 6.07 6.1 6.18 6.1 Nex-07 6.21 6.11 6.26 6.16 Nex-15 6.23 6.16 6.14 6.14 Nex-16 6.05 6.24 6.22 6.16 Nex-20 6.15 6.18 6.15 5.61 Nex-22 6.08 6.2 6.18 6.21 Nex-30 6.24 6.23 6.22 6.21 Nex-32 6.25 6.31 6.23 6.2 Nex-46 6.26 6.25 6.21 6.21 Nex-52 6.26 6.27 6.12 6.13 Nex-53 6.35 6.2 6.24 6.21 Nex-55 6.23 6.22 6.21 6.18 Nex-56 6.33 6.27 6.16 5.9 Nex-57 6.29 6.17 6.19 5.92 Nex-58 6.26 6.15 6.11 5.81 Nex-59 6.33 6.33 6.22 6.15 Nex-60 6.28 6.23 6.25 6.21 Nex-61 6.38 6.15 6.07 5.54 Nex-62 6.34 6.32 6.35 6.31 Nex-64 6.34 6.41 6.38 6.36 Nex-65 6.34 6.39 6.35 6.17 Nex-66 6.27 6.5 6.41 6.39 Nex-67 6.27 6.1 6.13 5.88 Nex-68 6.39 5.82 5.88 5.57 Nex-69 6.41 6.39 6.31 6.38 Nex-70 6.37 6.42 6.37 6.22 Nex-88 6.44 6.54 6.39 6.4

TABLE 7 Viable counts in Log₁₀ CFU/mL for Pseudomonas aeruginosa time kill study compound 0 0.5 min 1 min 5 min Nex-03 5.6 <4.0 <3.70 <3.52 Nex-05 5.64 <4.0 <3.70 <3.52 Nex-07 5.48 <4.0 <3.70 <3.52 Nex-16 5.64 4 3.7 <3.52 Nex-20 5.56 <4.0 3.7 <3.52 Nex-22 5.64 <4.0 <3.70 <3.52 Nex-30 5.67 <4.0 <3.70 <3.52 Nex-32 5.73 5.62 4.48 4.12 Nex-46 5.67 4.48 3.7 <3.52 Nex-52 5.56 4.7 4 <3.52 Nex-53 5.54 5.54 5.47 5.49 Nex-55 5.37 5.61 5.58 5.45 Nex-56 5.56 <4.0 <3.70 <3.52 Nex-57 5.45 <4.0 <3.70 <3.52 Nex-58 5.54 <4.0 <3.70 <3.52 Nex-59 5.65 5.26 4.78 <3.52 Nex-60 5.58 4.6 3.7 <3.52 Nex-61 5.71 4 <3.70 <3.52 Nex-62 5.52 4 <3.70 <3.52 Nex-64 5.45 4 <3.70 <3.52 Nex-65 5.52 5.36 5.52 <3.52 Nex-66 5.64 5.91 5.68 5.56 Nex-67 5.71 <4.0 <3.70 <3.52 Nex-68 5.78 5.18 4.88 4.87 Nex-69 5.65 5.75 5.74 5.21 Nex-70 5.62 <4.0 <3.70 <3.52 Nex-88 5.74 <4.0 <3.70 <3.52

TABLE 8 Viable counts in Log₁₀ CFU/mL for Candida albicans time kill study compound 0 0.5 min 1 min 5 min Nex-03 5.6 5.53 5.54 5.52 Nex-07 5.57 5.54 5.5 5.45 Nex-15 5.12 5.23 5.25 5.28 Nex-52 5.24 5.19 5.17 5.23 Nex-54 5.56 5.56 5.52 5.48 Nex-59 5.3 5.18 5.32 5.23 Nex-61 5.23 5.19 5.36 5.3 Nex-62 5.12 5.37 5.18 5.16 Nex-67 5.58 5.53 5.49 5.41 Nex-68 5.47 5.35 5.15 5.13 Nex-70 5.55 5.53 5.51 5.49

TABLE 9 Viable counts in Log₁₀ CFU/mL for Aspergillus niger time kill study compound 0 0.5 min 1 min 5 min Nex-52 5.30 5.28 5.28 4.95 Nex-59 5.27 5.30 5.33 5.18 Nex-61 5.36 5.30 5.16 5.11 Nex-62 5.27 5.32 5.21 5.02 Nex-68 5.25 5.25 5.25 5.25

Example 34 Extended Time Kill Studies

The time kill studies of Example 33 were extended for five compounds (Nex-59, Nex-60, Nex-61, Nex-62, and Nex-88) using the same methods at longer time periods and at higher multiples of MIC. Nex-88 is a code number for DDAIP.HCl from a different supplier. Time kill studies for five compounds were performed at 1×MIC, 2×MIC and 4×MIC concentration at six time points (0, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours). The viable counts in Log₁₀ CFU/mL for the five microorganisms were recorded, and are presented in Table 10 to Table 14 below.

Nex-59 Dodecyl 2- (methylamino) propanoate hydrochloride

Nex-60 Dodecyl 2- (isopropylamino) propanoate hydrochloride

Nex-61 Dodecyl 2-((2- hydroxyethyl) amino) propanoate hydrochloride

Nex-62 Dodecyl 2-((2- (diethylamino)- ethyl)amino propanoate dihydrochloride

Nex-05, Nex-88 DDAIP•HCl Dodecyl 2- (dimethylamino) propanoate hydrochloride

TABLE 10 Viable counts in Log₁₀ CFU/mL for Escherichia coli time kill study Compound 0 1 h 2 h 4 h 6 h 24 h NEX-59 1 × MIC 5 1 <1 1.85 2.81 7.91 (0.05 mg/mL) 2 × MIC 5.12 <1 <1 <1 <1 <1 (0.1 mg/mL) 4 × MIC 5.04 <1 <1 <1 <1 <1 (0.2 mg/mL) NEX-60 1 × MIC 5.02 <1 <1 <1 <1 <1 (3.13 mg/mL) 2 × MIC 5.14 <1 <1 <1 <1 <1 (6.25 mg/mL) 4 × MIC 5.07 <1 <1 <1 <1 <1 (12.5 mg/mL) NEX-61 1 × MIC 5.08 1.70 1.60 3.16 4.38 7.75 (0.78 mg/mL) 2 × MIC 5.03 <1 <1 <1 <1 <1 (1.56 mg/mL) 4 × MIC 5.05 <1 <1 <1 <1 <1 (3.13 mg/mL) NEX-62 1 × MIC 5.06 1 1 <1 2.18 7.99 (0.1 mg/mL) 2 × MIC 5.18 <1 <1 <1 <1 <1 (0.2 mg/mL) 4 × MIC 5.12 <1 <1 <1 <1 <1 (0.4 mg/mL) NEX-88 1 × MIC 5.10 <1 <1 <1 <1 <1 (3.13 mg/mL) 2 × MIC 5.16 <1 <1 <1 <1 <1 (6.25 mg/mL) 4 × MIC 5.21 <1 <1 <1 <1 <1 (12.5 mg/mL)

TABLE 11 Viable counts in Log₁₀ CFU/mL for Staphylococcus aureus time kill study Compound 0 1 h 2 h 4 h 6 h 24 h Nex-59 1 × MIC 6.20 <3 <3 <3 <3 <1 (0.05 mg/mL) 2 × MIC 6.19 <3 <3 <3 <3 <1 (0.1 mg/mL) 4 × MIC 6.18 <3 <3 <3 <3 <1 (0.2 mg/mL) Nex-60 1 × MIC 6.22 6.23 6.24 6.16 5.97 6.57 (1.56 mg/mL) 2 × MIC 6.20 <3 <3 <3 <3 <1 (3.13 mg/mL) 4 × MIC 6.21 <3 <3 <3 <3 <1 (6.25 mg/mL) Nex-61 1 × MIC 6.14 5 <3 <3 <3 <1 (0.05 mg/mL) 2 × MIC 6.20 <3 <3 <3 <3 <1 (0.1 mg/mL) 4 × MIC 6.18 <3 <3 <3 <3 <1 (0.2 mg/mL) Nex-62 1 × MIC 6.23 6.00 5.66 4.70 4.48 <1 (0.025 mg/mL) 2 × MIC 6.25 5.30 <3 <3 <3 <1 (0.05 mg/mL) 4 × MIC 6.20 5.30 <3 <3 <3 <1 (0.1 mg/mL) Nex-88 1 × MIC 6.24 <3 <3 <3 <3 <1 (3.13 mg/mL) 2 × MIC 6.25 <3 <3 <3 <3 <1 (6.25 mg/mL) 4 × MIC 6.23 <3 <3 <3 <3 <1 (12.5 mg/mL)

TABLE 12 Viable counts in Log¹⁰ CFU/mL for Pseudomonas aeruginosa time kill study Compound 0 1 h 2 h 4 h 6 h 24 h NEX-59 1 × MIC (3.13 mg/mL) 5.18 <1 <1 <1 <1 <1 2 × MIC (6.25 mg/mL) 5.16 <1 <1 <1 <1 <1 4 × MIC (12.5 mg/mL) 5.21 <1 <1 <1 <1 <1 NEX-60 1 × MIC (6.25 mg/mL) 5.30 <1 <1 <1 <1 <1 2 × MIC (12.5 mg/mL) 5.28 <1 <1 <1 <1 <1 4 × MIC (25.0 mg/mL) 5.31 <1 <1 <1 <1 <1 NEX-61 1 × MIC (6.25 mg/mL) 5.28 <1 <1 <1 <1 <1 2 × MIC (12.5 mg/mL) 5.29 <1 <1 <1 <1 <1 4 × MIC (25.0 mg/mL) 5.26 <1 <1 <1 <1 <1 NEX-62 1 × MIC (6.25 mg/mL) 5.20 <1 <1 <1 <1 <1 2 × MIC (12.5 mg/mL) 5.25 <1 <1 <1 <1 <1 4 × MIC (25.0 mg/mL) 5.23 <1 <1 <1 <1 <1 NEX-88 1 × MIC (6.25 mg/mL) 5.30 <1 <1 <1 <1 <1 2 × MIC (12.5 mg/mL) 5.22 <1 <1 <1 <1 <1 4 × MIC (25.0 mg/mL) 5.26 <1 <1 <1 <1 <1

TABLE 13 Viable counts in Log₁₀ CFU/mL for Candida albicans time kill study Compound 0 1 2 4 6 24 Nex-59 1 × MIC 5.68 5.41 5.24 4.96 4.40 >6 (0.025 mg/mL) 2 × MIC 5.75 3.48 3.00 3.00 <2.70 <1 (0.05 mg/mL) 4 × MIC 5.63 <3 <3 <2.70 <2.70 <1 (0.1 mg/mL) Nex-60 1 × MIC 5.56 5.51 5.50 5.18 5.09 >6 (12.5 mg/mL) 2 × MIC 5.51 3.30 3.00 3.00 <2.70 <1 (25.0 mg/mL) 4 × MIC 5.62 3.78 3.30 3.00 <2.70 <1 (50.0 mg/mL) Nex-61 1 × MIC 5.43 5.37 5.37 5.32 5.54 >6 (0.0125 mg/mL) 2 × MIC 5.48 <3 <3 <2.70 <2.70 >6 (0.025 mg/mL) 4 × MIC 5.39 <3 <3 <2.70 <2.70 <1 (0.05 mg/mL) Nex-62 1 × MIC 5.74 5.75 5.78 6.04 6.21 >6 (0.00625 mg/mL) 2 × MIC 5.76 5.68 5.67 5.68 5.58 >6 (0.0125 mg/mL) 4 × MIC 5.69 5.32 5.33 5.00 4.53 <1 (0.025 mg/mL) Nex-88 1 × MIC 5.82 5.62 5.60 5.60 6.02 >6 (>12.5 mg/mL) 2 × MIC 5.81 4.99 4.88 4.62 4.58 2.57 (25.0 mg/mL) 4 × MIC 5.79 3.95 3.90 <2.70 <2.70 <1 (50 mg/mL)

TABLE 14 Viable counts in Log₁₀ CFU/mL for Aspergillus niger time kill study Compound 0 h 1 h 2 h 4 h 6 h 24 h NEX-59 1 × MIC 5.41 5.34 5.48 5.08 5.30 3.78 (0.05 mg/mL) 2 × MIC 5.36 5.11 4.70 4.48 3.95 <2 (0.1 mg/mL) 4 × MIC 5.29 5.08 4.48 3.85 4.04 <2 (0.2 mg/mL) NEX-60 1 × MIC 5.52 4.60 4.78 4.60 4.30 3.81 (12.5 mg/mL) 2 × MIC 5.47 4.30 4.78 3.85 3.78 4.30 (25.0 mg/mL) 4 × MIC 5.60 4.00 4.48 3.60 3 <2 (50.0 mg/mL) NEX-61 1 × MIC 5.28 5.48 5.36 5.18 4.95 4.48 (0.025 mg/mL) 2 × MIC 5.33 5.52 5.57 4.48 4.90 3 (0.05 mg/mL) 4 × MIC 5.26 5.34 5.41 5.28 4.95 <2 (0.1 mg/mL) NEX-62 1 × MIC 5.51 5.53 5.51 5.15 4.78 4 (0.0125 mg/mL) 2 × MIC 5.49 5.48 5.57 5.08 5.38 5.58 (0.025 mg/mL) 4 × MIC 5.56 5.49 5.49 5.00 5.20 3.85 (0.05 mg/mL) NEX-88 1 × MIC 5.41 5.28 4.78 4.60 4.30 3.30 (12.5 mg/mL) 2 × MIC 5.38 4.30 4.70 3 4 <2 (25.0 mg/mL) 4 × MIC 5.50 4.48 4 3 4 <2 (50.0 mg/mL)

Example 35 Minimum Inhibitory Concentration Assays with Different Strains of Microorganisms

The minimum inhibitory concentration (MIC) is used to determine the lowest concentration of a test product claiming antimicrobial effects that will inhibit growth of a microorganism. Each microbial suspension was adjusted approximately 10⁷ to 10⁸ colony forming units (CFU) per mL and labeled as the stock suspension. The stock suspension was further diluted to 1:200 in Mueller Hinton broth to obtain a test sample having a concentration of 10¹ to 10⁶ CFU/mL. The test sample was then tested for MIC as described in M202.R02. After incubation, each tube was examined for turbidity, which indicated growth or no growth. For the tubes where the product rendered the media turbid, an aliquot of the media was streaked onto agar plates to confirm growth or lack of growth. The results are presented in Tables 15-18, below.

TABLE 15 DDAIP•HCl MIC Sample Concentration 10% 5% 2.5% 1.25% 0.625% 0.313% 0.156% 0.078% 0.039% Control MIC Escherichia coli NG NG NG NG NG Growth Growth Growth Growth Growth 0.625% ATCC 11229 Pseudomonas NG NG NG NG NG Growth Growth Growth Growth Growth  1.25% aeruginosa ATCC 9027 Staphylococcus NG NG NG Growth Growth Growth Growth Growth Growth Growth  2.5% aureus ATCC 6538 Bacillus subtilis NG NG NG NG NG NG Growth Growth Growth Growth 0.313% ATCC# 6633 Candida albicans NG NG NG NG NG NG Growth Growth Growth Growth 0.313% ATCC 10231 Acinetobacter NG NG NG NG NG NG Growth Growth Growth Growth 0.313% baumannii ATCC 19606 Burkholderia NG NG NG Growth Growth Growth Growth Growth Growth Growth  2.5% cepacia ATCC 25416 Enterococcus NG NG NG NG NG NG Growth Growth Growth Growth 0.313% faecalis ATCC 51299 Klebsiella NG NG NG NG NG NG Growth Growth Growth Growth 0.625% pneumoniae ATCC# 11296 Staphylococcus NG NG NG NG NG Growth Growth Growth Growth Growth 0.625% aureus (MRSA) ATCC 33592 Staphylococcus NG NG NG NG NG NG Growth Growth Growth Growth 0.313% haemolyticus ATCC 29970

TABLE 16 DDAIP•HCl MIC Sample Concentration 10% 5% 2.5% 1.25% 0.625% 0.313% 0.156% 0.078% 0.039% Control MIC Enterobacter NG NG NG NG NG Growth Growth Growth Growth Growth 0.625% cloacae ATCC 13047 Proteus mirabilis NG NG NG NG Growth Growth Growth Growth Growth Growth 1.25% ATCC 7002 Micrococcus NG NG NG NG NG NG Growth Growth Growth Growth 0.313% luteus ATCC 4698 Enterobacter NG NG NG NG Growth Growth Growth Growth Growth Growth  1.25% aerogenes ATCC 13048 Streptococcus NG NG NG NG NG NG Growth Growth Growth Growth 0.313% pyogenes ATCC 19615 Escherichia coli NG NG NG NG Growth Growth Growth Growth Growth Growth  1.25% ATCC 25922 Klebsiella NG NG NG NG Growth Growth Growth Growth Growth Growth  1.25% oxytoca ATCC 43165 Serratia NG NG NG Growth Growth Growth Growth Growth Growth Growth  2.5% marcescens ATCC 14756 Staphylococcus NG NG NG NG NG Growth Growth Growth Growth Growth 0.625% saprophyticus ATCC 15305 Corynebacterium NG NG NG NG NG Growth Growth Growth Growth Growth 0.625% jeikeium ATCC 43734 Enterococcus NG NG NG NG Growth Growth Growth Growth Growth Growth  1.25% faecalis ATCC 19433 Staphylococcus NG NG NG NG NG NG Growth Growth Growth Growth 0.313% epidermidis ATCC 12228 Staphylococcus NG NG NG NG NG Growth Growth Growth Growth Growth 0.625% hominis ATCC 27844 Streptococcus NG NG NG NG NG NG NG Growth Growth Growth 0.156% pneumoniae ATCC 6303 Acinetobacter NG NG NG NG NG NG NG Growth Growth Growth 0.156% iwoffi ATCC 15309 Haemophilus NG NG Growth Growth Growth Growth Growth Growth Growth Growth    5% influenzae ATCC 19418

TABLE 17 Nex-59 MIC Sample Concentration 2.5% 1.25% 0.63% 0.32% 0.16% 0.08% 0.04% 0.02% 0.01% Control MIC Burkholderia NG Growth Growth Growth Growth Growth Growth Growth Growth Growth  2.5% cepacia ATCC 25416 Enterobacter NG NG NG NG NG Growth Growth Growth Growth Growth 0.16% cloacae ATCC 13047 Escherichia coli NG NG NG NG NG NG Growth Growth Growth Growth 0.08% ATCC 11229 Proteus mirabilis NG NG NG NG Growth Growth Growth Growth Growth Growth 0.32% ATCC 7002 Pseudomonas NG NG NG NG NG Growth Growth Growth Growth Growth 0.16% aeruginosa ATCC 9027 Bacillus subtilis NG NG NG NG NG NG NG Growth Growth Growth 0.04% ATCC 6633 Staphylococcus NG NG NG NG NG NG NG Growth Growth Growth 0.04% aureus ATCC 6538 Candida NG NG NG NG NG NG NG Growth Growth Growth 0.04% albicans ATCC 10231 Acinetobacter NG NG NG NG NG NG Growth Growth Growth Growth 0.08% baumannii ATCC19606 Enterococcus NG NG NG NG NG NG NG Growth Growth Growth 0.04% faecium ATCC 19434 Acinetobacter NG NG NG NG NG NG NG NG Growth Growth 0.02% iwoffi ATCC4 15309 Streptococcus NG NG NG NG Growth Growth Growth Growth Growth Growth 0.32% pneumoniae ATCC 6303

TABLE 18 Nex-59 MIC Sample Concentration 2.5% 1.25% 0.63% 0.32% 0.16% 0.08% 0.04% 0.02% 0.01% Control MIC Klebsiella No No No No No No Growth Growth Growth Growth 0.08% pneumoniae Growth Growth Growth Growth Growth Growth ATCC 11296 Enterococcus No No No No No No No Growth Growth Growth 0.04% faecalis Growth Growth Growth Growth Growth Growth Growth ATCC 51299 Enterobacter No No No No No Growth Growth Growth Growth Growth 0.16% aerogenes Growth Growth Growth Growth Growth ATCC 13048 Micrococcus No No No No No No No No Growth Growth 0.02% luteus Growth Growth Growth Growth Growth Growth Growth Growth ATCC 4698 Staphylococcus No No No No No No No No Growth Growth 0.02% epidermidis Growth Growth Growth Growth Growth Growth Growth Growth ATCC 12228 Staphylococcus No No No No No No No No Growth Growth 0.02% haemolyticus Growth Growth Growth Growth Growth Growth Growth Growth ATCC 29970 Staphylococcus No No No No No No No No Growth Growth 0.02% hominis Growth Growth Growth Growth Growth Growth Growth Growth ATCC 27844 Staphylococcus No No No No No No No No Growth Growth 0.02% saprophyticus Growth Growth Growth Growth Growth Growth Growth Growth ATCC 15305 Staphylococcus No No No No No No No No Growth Growth 0.02% aureus (MRSA) Growth Growth Growth Growth Growth Growth Growth Growth ATCC 33592 Haemophilus No No No No No No No No No Growth ≦0.01%   influenzae Growth Growth Growth Growth Growth Growth Growth Growth Growth ATCC 19418 Streptococcus No No No No No No No No No Growth ≦0.01%   pyogenes Growth Growth Growth Growth Growth Growth Growth Growth Growth ATCC 19615 Corynebacterium No No No No No No No No No Growth ≦0.01%   jeikeium Growth Growth Growth Growth Growth Growth Growth Growth Growth ATCC 43734 Enterococcus No No No No No No No No No Growth ≦0.01%   faecalis Growth Growth Growth Growth Growth Growth Growth Growth Growth ATCC 19433 Escherichia coli No No No No No Growth Growth Growth Growth Growth 0.16% ATCC 25922 Growth Growth Growth Growth Growth Klebsiella No No No Growth Growth Growth Growth Growth Growth Growth 0.63% oxytoca Growth Growth Growth ATCC 43I65 Serratia Growth Growth Growth Growth Growth Growth Growth Growth Growth Growth >2.5% marcescens ATCC 14756

TABLE 19 MIC (mg/ml) Nex-01 Nex-03 Nex-05 Nex-07 Nex-15 Nex-16 Acinetobacter baumannii (ATCC 19606) 3.13 Acinetobacter iwoffi (ATCC 15309) 1.56 Aspergillus niger (CMCC 98003) >12.5 >12.5 >12.5 12.5 12.5 >12.5 Bacillus subtilis (ATCC 6633) 3.13 Burkholderia cepacia (ATCC 25416) 25 Candida albicans (ATCC 10231) 3.13 Candida albicans (ATCC 90029) >12.5 12.5, >12.5 12.5, 0.05 12.5 6.25 6.25 Corynebacterium jeikeium (ATCC 43734) 6.25 Enterobacter aerogenes (ATCC 13048) 12.5 Enterobacter cloacae (ATCC 13047) 6.25 Enterococcus faecalis (ATCC 51299) 3.13 Enterococcus faecalis (ATCC 19433) 12.5 Enterococcus faecium (ATCC 19434) Escherichia coli (ATCC 11229) 6.25 Escherichia coli (ATCC 25922) 3.13 1.56 3.13 0.78, ≧12.5 3.13 1.56, 12.5 Haemophilus influenza (ATCC 19418) 50 Klebsiella oxytoca (ATCC 43615) 12.5 Klebsiella pneumonia (ATCC 11296) 6.25 Micrococcus luteus (ATCC 4698) 3.13 Proteus mirabilis (ATCC 7002) 12.5 Pseudomonas aeruginosa (ATCC 9027) 12.5 Pseudomonas aeruginosa (ATCC 27853) >12.5 3.13 6.25 3.13 >12.5 12.5 Serratia marcescens (ATCC 14756) 25 Staphylococcus aureus (ATCC 6538) 25 Staphylococcus aureus (MRSA) (ATCC 6.25 33592) Staphylococcus aureus (ATCC 29213) 3.13 1.56 3.13, 1.56, 0.39, 3.13 1.56 0.78 0.2 Staphylococcus epidermidis (ATCC 12228) 3.13 Staphylococcus haemolyticus (ATCC 29970) 3.13 Staphylococcus hominis (ATCC 27844) 6.25 Streptococcus pneumonia (ATCC 6303) 1.56 Staphylococcus saprophyticus (ATCC 6.25 15305) Streptococcus pyogenes (ATCC 19615) 3.13 Nex-20 Nex-22 Nex-30 Nex-32 Nex-46 Nex-52 Acinetobacter baumannii (ATCC 19606) Acinetobacter iwoffi (ATCC 15309) Aspergillus niger (CMCC 98003) >12.5 >12.5 >12.5 >12.5 >12.5 3.13 Bacillus subtilis (ATCC 6633) Burkholderia cepacia (ATCC 25416) Candida albicans (ATCC 10231) Candida albicans (ATCC 90029) 12.5 >12.5 >12.5 >12.5 >12.5 0.05 Corynebacterium jeikeium (ATCC 43734) Enterobacter aerogenes (ATCC 13048) Enterobacter cloacae (ATCC 13047) Enterococcus faecalis (ATCC 51299) Enterococcus faecalis (ATCC 19433) Enterococcus faecium (ATCC 19434) Escherichia coli (ATCC 11229) Escherichia coli (ATCC 25922) 1.56 3.13 3.13 3.13 3.13 1.56 Haemophilus influenza (ATCC 19418) Klebsiella oxytoca (ATCC 43615) Klebsiella pneumonia (ATCC 11296) Micrococcus luteus (ATCC 4698) Proteus mirabilis (ATCC 7002) Pseudomonas aeruginosa (ATCC 9027) Pseudomonas aeruginosa (ATCC 27853) 6.25 6.25 6.25 6.25 6.25 6.25 Serratia marcescens (ATCC 14756) Staphylococcus aureus (ATCC 6538) Staphylococcus aureus (MRSA) (ATCC 33592) Staphylococcus aureus (ATCC 29213) 3.13, 3.13 3.13 1.56 3.13 0.2, 0.1 1.56 Staphylococcus epidermidis (ATCC 12228) Staphylococcus haemolyticus (ATCC 29970) Staphylococcus hominis (ATCC 27844) Streptococcus pneumonia (ATCC 6303) Staphylococcus saprophyticus (ATCC 15305) Streptococcus pyogenes (ATCC 19615) Nex-53 Nex-54 Nex-55 Nex-56 Nex-57 Nex-58 Acinetobacter baumannii (ATCC 19606) Acinetobacter iwoffi (ATCC 15309) Aspergillus niger (CMCC 98003) >12.5 12.5 >12.5 >12.5 >12.5 >12.5 Bacillus subtilis (ATCC 6633) Burkholderia cepacia (ATCC 25416) Candida albicans (ATCC 10231) Candida albicans (ATCC 90029) >12.5 6.25 >12.5 >12.5 >12.5 >12.5 Corynebacterium jeikeium (ATCC 43734) Enterobacter aerogenes (ATCC 13048) Enterobacter cloacae (ATCC 13047) Enterococcus faecalis (ATCC 51299) Enterococcus faecalis (ATCC 19433) Enterococcus faecium (ATCC 19434) Escherichia coli (ATCC 11229) Escherichia coli (ATCC 25922) 6.25 ≧12.5 6.25 3.13 3.13 3.13 Haemophilus influenza (ATCC 19418) Klebsiella oxytoca (ATCC 43615) Klebsiella pneumonia (ATCC 11296) Micrococcus luteus (ATCC 4698) Proteus mirabilis (ATCC 7002) Pseudomonas aeruginosa (ATCC 9027) Pseudomonas aeruginosa (ATCC 27853) 6.25 ≧12.5 6.25 6.25 6.25 6.25 Serratia marcescens (ATCC 14756) Staphylococcus aureus (ATCC 6538) Staphylococcus aureus (MRSA) (ATCC 33592) Staphylococcus aureus (ATCC 29213) 6.25 ≧12.5 6.25 3.13 3.13 3.13, 1.56 Staphylococcus epidermidis (ATCC 12228) Staphylococcus haemolyticus (ATCC 29970) Staphylococcus hominis (ATCC 27844) Streptococcus pneumonia (ATCC 6303) Staphylococcus saprophyticus (ATCC 15305) Streptococcus pyogenes (ATCC 19615) Nex-59 Nex-60 Nex-61 Nex-62 Nex-64 Nex-65 Acinetobacter baumannii (ATCC 19606) 0.8 Acinetobacter iwoffi (ATCC 15309) 0.2 Aspergillus niger (CMCC 98003) 0.05 >12.5 0.05, 0.0125 >12.5 >12.5 0.025 Bacillus subtilis (ATCC 6633) 0.4 Burkholderia cepacia (ATCC 25416) 25 Candida albicans (ATCC 10231) 0.4 Candida albicans (ATCC 90029) 0.05 12.5 0.0125 0.00625 >12.5 >12.5 Corynebacterium jeikeium (ATCC 43734) ≦0.1 Enterobacter aerogenes (ATCC 13048) 1.6 Enterobacter cloacae (ATCC 13047) 1.6 Enterococcus faecalis (ATCC 51299) 0.4 Enterococcus faecalis (ATCC 19433) ≦0.1 Enterococcus faecium (ATCC 19434) 0.4 Escherichia coli (ATCC 11229) 0.8 Escherichia coli (ATCC 25922) 0.05, 1.6 3.13 0.78, 0.1 3.13 3.13 1.56 Haemophilus influenza (ATCC 19418) ≦1 Klebsiella oxytoca (ATCC 43615) 6.3 Klebsiella pneumonia (ATCC 11296) 0.8 Micrococcus luteus (ATCC 4698) 0.2 Proteus mirabilis (ATCC 7002) 3.2 Pseudomonas aeruginosa (ATCC 9027) 1.6 Pseudomonas aeruginosa (ATCC 27853) 3.13 6.25 6.25 6.25 6.25 6.25 Serratia marcescens (ATCC 14756) >25 Staphylococcus aureus (ATCC 6538) 0.4 Staphylococcus aureus (MRSA) (ATCC 0.2 33592) Staphylococcus aureus (ATCC 29213) 0.05 1.56 0.05 0.025 3.13 3.13 Staphylococcus epidermidis (ATCC 12228) 0.2 Staphylococcus haemolyticus (ATCC 29970) 0.2 Staphylococcus hominis (ATCC 27844) 0.2 Streptococcus pneumonia (ATCC 6303) 3.2 Staphylococcus saprophyticus (ATCC 15305) 0.2 Streptococcus pyogenes (ATCC 19615) ≦0.1 Nex-66 Nex-67 Nex-68 Nex-69 Nex-70 Nex-88 Acinetobacter baumannii (ATCC 19606) Acinetobacter iwoffi (ATCC 15309) Aspergillus niger (CMCC 98003) >12.5 12.5 3.13 >12.5 12.5 >12.5 Bacillus subtilis (ATCC 6633) Burkholderia cepacia (ATCC 25416) Candida albicans (ATCC 10231) Candida albicans (ATCC 90029) >12.5 6.25 0.39, 0.2 12.5 0.78 >12.5 Corynebacterium jeikeium (ATCC 43734) Enterobacter aerogenes (ATCC 13048) Enterobacter cloacae (ATCC 13047) Enterococcus faecalis (ATCC 51299) Enterococcus faecalis (ATCC 19433) Enterococcus faecium (ATCC 19434) Escherichia coli (ATCC 11229) Escherichia coli (ATCC 25922) 3.13 1.56 0.78, 3.13 0.78 3.13 1.56 Haemophilus influenza (ATCC 19418) Klebsiella oxytoca (ATCC 43615) Klebsiella pneumonia (ATCC 11296) Micrococcus luteus (ATCC 4698) Proteus mirabilis (ATCC 7002) Pseudomonas aeruginosa (ATCC 9027) Pseudomonas aeruginosa (ATCC 27853) 6.25 6.25 3.13 6.25 3.13 6.25 Serratia marcescens (ATCC 14756) Staphylococcus aureus (ATCC 6538) Staphylococcus aureus (MRSA) (ATCC 33592) Staphylococcus aureus (ATCC 29213) 3.13 3.13, 3.13 3.13 3.13 3.13 1.56 Staphylococcus epidermidis (ATCC 12228) Staphylococcus haemolyticus (ATCC 29970) Staphylococcus hominis (ATCC 27844) Streptococcus pneumonia (ATCC 6303) Staphylococcus saprophyticus (ATCC 15305) Streptococcus pyogenes (ATCC 19615)

Table 19, above, is a summary of the MIC results of Example 33 and Example 35 for all tested antimicrobial compounds and all tested strains.

While the disclosure has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof. Therefore, it is intended that the disclosure not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all embodiments falling within the scope of the appended claims. 

1. A compound of Formula I:

where n is an integer from 6-12 inclusive; R¹, and R², are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, CH₃, CHOHCH₃, CH(CH₃)₂, CH₂C₆H₆, and CH₂CH(CH₃)₂; R³, and R⁴, are the same or different and are selected independently from the group consisting of H, substituted or unsubstituted, straight or branched chain C₁-C₁₀ alkyl, C₆-C₁₀ aryl or C₆-C₁₀ heteroaryl, CH₃, CH(CH₃)₂, CH₂CH₂OH, CH₂CH(CH₃)₂, and (CH₂)₂N(CH₂CH₃)₂; R⁵ is methyl, and R⁶ is H or CH₃, and pharmaceutically acceptable salts thereof.
 2. A composition comprising the compound of claim 1 and a pharmaceutically acceptable excipient.
 3. The composition of claim 2 further comprising an active pharmaceutical ingredient.
 4. The composition of claim 2 further comprising an additive.
 5. The composition of claim 4 wherein the additive is at least one of a fragrance, an emulsifier, a stabilizer and a preservative.
 6. The composition of claim 2 wherein the composition is provided in an unit dosage form.
 7. The composition of claim 6 wherein the unit dosage form is a tablet, a pill, a capsule, a powder, a granule, a sterile parenteral solution or a sterile parenteral suspension, a metered aerosol spray, a metered liquid spray, a drop, an ampoule, an auto-injector device, or a suppository.
 8. The composition of claim 7 formulated for inhalation, oral, parenteral, intranasal, sublingual or rectal administration. 